Cbln1 is one of the most recently identified bidirectional synaptic organizers in the cerebellum; Cbln1 secreted from cerebellar granule cells indirectly serves as a postsynaptic organizer by binding to its postsynaptic receptor GluD2 expressed in Purkinje cells and directly induces presynaptic differentiation (Matsuda et al., 2010). However, it remained unclear how Cbln1 binds to the presynaptic sites and interacts with other synaptic organizers. In this study, we found that Cbln1 competed with synaptogenesis mediated by NL-NRX and identified NRX1α(S4+) and NRXβs(S4+) as presynaptic receptors for Cbln1. While this manuscript was in preparation, Uemura et al. (2010) also reported the interaction of Cbln1 with NRXs in the cerebellum. We further showed that not only Cbln1, but also its family member Cbln2 but not Cbln4 specifically bound to NRX1β(S4+) even under low Ca2+-concentrations, which was distinct from the interaction between NRXs and NLs or NRXs and LRRTM2. We also characterized in detail the nature of the tripartite complex NRXs/Cbln1/GluD2 as a bidirectional organizer. Finally, we showed that Cbln members induced synaptogenesis in hippocampal and cortical neurons as well as in the cerebellar neurons (Fig. 7 and Supporting Information Fig. S5).
Unique synaptic signaling by the tripartite complex neurexin/Cbln1/GluD2
The cell-based (Fig. 2) and in vitro (Fig. 6) binding assays showed that NRX1α and NRX1–3β carrying the splice site 4 insert specifically bound to Cbln1. Cbln1 coated on beads directly accumulated NRX1β(S4+) on granule cell axons (Fig. 4B and Supporting Information Fig. S2A) and Cbln1-induced presynaptic differentiation was specifically inhibited by soluble NRX1β(S4+)-Fc (Fig. 4C), indicating that NRXs(S4+) serves as a presynaptic receptor for Cbln1. In addition, NRX1β(S4+) coated on beads clustered GluD2 and its interacting intracellular protein shank2 in postsynaptic Purkinje cells in a Cbln1-dependent manner (Fig. 5B). These results indicate that the tripartite complex consisting of NRX(S4+), Cbln1 and GluD2 could serve as a bidirectional synaptic organizer.
The NRX/Cbln1/GluD2 complex has several unique features as a synapse organizer (Fig. 8). First, unlike NRXs/NLs (Nguyen & Sudhof, 1997) or NRXs/LRRTMs (Ko et al., 2009; Siddiqui et al., 2010), this complex was resistant to low extracellular Ca2+ concentrations. The crystal structure of NRX1β indicates that Ca2+ binding is essential for binding to NLs (Koehnke et al., 2008). Similarly, other NRX ligands, such as LRRTMs and α-dystroglycan (Sugita et al., 2001), also bind to NRX in a Ca2+-dependent manner. In contrast, neurexophilins bind to the second laminin, NRX, sex-hormone-binding protein (LNS) domain in NRXα in a Ca2+-independent manner (Missler et al., 1998). Unlike neurexophilins but like NLs and LRRTMs, Cbln1 binds to both NRXα and NRXβ, suggesting that Cbln1 binds to the sixth LNS domain in which the splice site 4 insert is located (Craig & Kang, 2007). Structural studies on NRX1β(S4+) have shown that the splice site 4 insert is unstructured and remains partially disordered in the complex with NLs despite its high level of sequence conservation, suggesting that it has a distinct functional role in binding to partner molecules other than NLs (Koehnke et al., 2008). Together, these findings indicate that Cbln1 binds to the region involving the splice site 4 insert of NRXs in a manner distinct from NLs or LRRTMs. Although it remains unclear whether Cbln1 and NLs compete for presynaptic NRXs in vivo, Cbln1 inhibited the interaction between NL1(−) and NRX(S4+) in vitro (Fig. 1) probably by steric hindrance because Cbln1 and NL1(−) are unlikely to share the same binding site of NRX(S4+).
Figure 8. Schematic drawing summarizing a unique synaptic signaling by the tripartite complex NRX/Cbln1/GluD2. Cbln1 serves as a bidirectional synaptic organizer by binding to presynaptic NRXs(S4+) and postsynaptic GluD2 in hippocampal, cortical and cerebellar neurons. Cbln2 but not Cbln4 is likely to have a similar synaptogenic function. Although LRRTMs and NLs bind to NRXs in a Ca2+-dependent manner, the NRX/Cbln1/GluD2 complex is maintained under low Ca2+ concentration. In contrast to LRRTMs, which bind to NRXs(S4−) and mediate excitatory synaptogenesis, Cbln1/GluD2 specifically binds to NRXs(S4+) and regulates excitatory and inhibitory synaptogenesis, depending on the type of neurons expressing Cbln proteins. NMDAR, N-methyl-D-aspartate receptor; PSD95, postsynaptic density 95; VGluT, vesicular glutamate transporter; VGAT, vesicular GABA transporter.
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Although various cell adhesion molecules (such as cadherins, protocadherins, NRXs/NLs and NRXs/LRRTMs) require extracellular Ca2+, synaptic adhesion itself is independent of Ca2+ (Sudhof, 2001). cbln1- and GluD2-null mice are ataxic, showing a markedly impaired performance on the rotorod test. Although their cerebellum appears grossly normal, detailed electrophysiological and electron microscopic analyses of these mice revealed that the number of PF–Purkinje cell synapses is markedly reduced, and most dendritic spines have lost synaptic contact with the PFs. In addition, in the remaining PF–Purkinje cell synapses, the postsynaptic densities are disproportionally longer than the presynaptic active zones. These unique morphological phenotypes and Ca2+-resistant binding of the NRX/Cbln1/GluD2 complex is consistent with the function of the complex as synaptic glue, connecting pre- and postsynaptic elements.
The second unique feature of the NRX/Cbln1/GluD2 complex is that the secreted Cbln1 works by being sandwiched between presynaptic NRX and postsynaptic GluD2. In central nervous system synapses, synaptic organizers are classified into two categories: cell adhesion molecules that directly link pre- and postsynaptic elements and soluble factors. Most soluble synaptic organizers in the central nervous system, such as neuronal pentraxins (Xu et al., 2003), fibroblast growth factors (Terauchi et al., 2010) and Wnt-7a (Hall et al., 2000), work on either the pre- or postsynaptic site, depending on the location of their receptors (Johnson-Venkatesh & Umemori, 2010). Thus, the sandwich-type signaling by the NRX/Cbln1/GluD2 complex is unique in that secreted Cbln1 serves as a bidirectional synaptic organizer. For Cbln1 to bind to pre- and postsynaptic receptors simultaneously, Cbln1 needs to have at least two binding sites. This could have been achieved by the presence of multiple binding sites within single Cbln1 monomers or by the presentation of single binding sites in different directions by forming a multimeric Cbln1 complex (Iijima et al., 2007). Recently, glial-derived neurotrophic factor was also proposed to serve as a synaptic adhesion molecule being sandwiched by its receptor glial-derived neurotrophic factor family receptor (GFR)α1 located at pre- and postsynaptic neurons (Ledda et al., 2007). In addition, leucine-rich glioma inactivated 1 was recently shown to be secreted from neurons and to organize presynaptic potassium channels and postsynaptic AMPA receptors by binding to its pre- and postsynaptic receptors, a disintegrin and metalloproteinase (ADAM) 22 and ADAM23, respectively (Fukata et al., 2010). These recent findings indicate that the sandwich type constitutes the third category of synaptic organizers.
Advantages of sandwich-type synaptic organizers may include an additional level of regulation of synapse formation and its functions. For example, the expression of cbln1 mRNA is completely shut down in granule cells when neuronal activity is increased for several hours (Iijima et al., 2009). Similarly, a sustained increase in neuronal activity causes the internalization of GluD2 from the postsynaptic site of cultured Purkinje cells (Hirai, 2001). As Cbln1 and NLs compete for NRXs, such activity-dependent regulation of Cbln1 and GluD2 might lead to switching between NRX/NL and NRX/Cbln1/GluD2 modes of synaptogenesis. Furthermore, the splicing of site 4 of NRXs was also shown to be regulated during development and in response to neurotrophic factors in chicken (Patzke & Ernsberger, 2000) and by ischemia in rats (Sun et al., 2000). Thus, each component of the NRX/Cbln1/GluD2 complex may be differentially regulated at the transcriptional and post-translational levels and such fine tuning of the NRX/Cbln1/GluD2 complex may play a role in the structural changes observed at PF synapses following increased neuronal activity in the adult cerebellum (Black et al., 1990).
Cbln1 and Cbln2 serve as synaptic organizers in various brain regions
Cbln1 mRNA is highly expressed in the cerebellum, but it is also enriched in a subset of neurons in various brain regions, including the mitral layer of the olfactory bulb, retrosplenial granular cortex, entorhinal cortex and thalamic parafascicular nucleus (Miura et al., 2006). Nevertheless, it is unclear whether Cbln1 is involved in synaptogenesis in these brain regions. We showed that Cbln1-coated beads were capable of inducing hemisynaptic differentiation of hippocampal and cortical neurons in vitro. Interestingly, in cbln1-null mice the spine density of medial spiny neurons in the striatum, which receive inputs from the Cbln1-positive thalamic parafascicular nucleus, was markedly increased, suggesting that Cbln1 determines the dendritic structure of striatal neurons with effects distinct from those seen in the cerebellum (Kusnoor et al., 2010). Although GluD2 is not expressed, its family member GluD1, which also binds to HA-Cbln1 (Matsuda et al., 2010), is highly expressed in these brain regions, especially during development (Lomeli et al., 1993). Therefore, a possible explanation for this difference is that GluD1 may mediate postsynaptic effects distinct from those regulated by GluD2. Indeed, Cbln1-coated beads did not accumulate AMPA receptors in hippocampal neurons (Supporting Information Fig. S4B) although endogenous GluD1 is expressed in these neurons (data not shown), suggesting that, unlike GluD2, GluD1 may not associate with scaffolding proteins such as shank2. Further studies are required to determine the signaling pathways regulated by Cbln1 outside the cerebellum.
The Cbln family consists of four members, Cbln1–Cbln4. Although Cbln3 is specifically expressed in cerebellar granule cells, other members are expressed in various brain regions (Miura et al., 2006). We showed that Cbln1 and Cbln2 but not Cbln4 were capable of binding to NRX1β(S4+) and inducing hemisynaptic differentiation of cerebellar, hippocampal and cortical neurons in vitro. Such differential effects were rather unexpected, as the amino acid sequences of the coding regions of Cbln1, Cbln2 and Cbln4 are very similar to each other (87–91%) (Yuzaki, 2008). As Cbln4 is always coexpressed with Cbln1 or Cbln2 in most brain regions (Miura et al., 2006), such as the entorhinal cortex and thalamic parafascicular nucleus, Cbln4 may serve as a synaptic organizer by forming a heteromer complex (Fig. 7C), and possibly by modulating the synaptogenic activities of Cbln1 and Cbln2. Future studies using gene knockout mice are required to elucidate the synaptic roles of Cbln and GluD family proteins in the central nervous system.