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Cbln1 and its family proteins in synapse formation and maintenance
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Cbln1 and its family proteins in synapse formation and maintenance Michisuke Yuzaki Cbln1 is a newly identified synaptic organizer belonging to the C1q family. Unlike other synaptic organizers, a deficiency in Cbln1 is sufficient to cause a severe reduction in the number of synapses between cerebellar Purkinje cells and parallel fibers (PFs). Furthermore, Cbln1 can rapidly induce synaptogenesis and is necessary for maintaining normal synapses in the mature cerebellum in vivo. Cbln1 was recently identified as the missing ligand for the orphan glutamate receptor d2 (GluD2), which is expressed in Purkinje cells. Furthermore, Cbln1 released from PFs binds to neurexin (NRX) expressed on the presynaptic PFs and GluD2 at the postsynaptic site. The NRX/Cbln1/GluD2 tripartite complex is resistant to low extracellular Ca2+ levels and serves as a unique bidirectional synaptic organizer. Address Department of Neurophysiology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan Corresponding author: Yuzaki, Michisuke ([email protected])
Cbln1 is a newly identified synaptic organizer released from granule cells in the cerebellum [10]. Cbln1 belongs to the C1q family [11], most of which are secreted (e.g. the complement C1q and adipokine adiponectin), and play diverse roles in intercellular communication [12]. Unlike other synaptic organizers, a deficiency in Cbln1 alone is sufficient to cause a severe reduction in the number of synapses between Purkinje cells and parallel fibers (PFs; axons of granule cells), which results in cerebellar ataxia in mice [10]. A single injection of recombinant Cbln1 into the subarachnoid supracerebellar space rapidly restores PF–Purkinje cell synapses and rescues the ataxia in adult cbln1-null mice within a day, but the effect is transient, and the synapses are lost when the injected Cbln1 is degraded [13]. Similarly, ablation of Cbln1 in the adult cerebellum resulted in a severe reduction in the number of PF– Purkinje cell synapses [14]. Therefore, Cbln1 is a unique synaptic organizer that can rapidly induce synaptogenesis and is necessary for maintaining normal synapses in the mature brain in vivo.
Current Opinion in Neurobiology 2011, 21:215–220 This review comes from a themed issue on Synaptic function and regulation Edited by Yukiko Goda and Bernardo Sabatini Available online 20th February 2011 0959-4388/$ – see front matter # 2011 Elsevier Ltd. All rights reserved.
Recently, two receptors for Cbln1 were identified. One, NRX, is located at the presynaptic site of PF–Purkinje cell synapses; the other, glutamate receptor d2 (GluD2), is postsynaptic [14,15]. In this review, I will summarize the most recent findings regarding NRX/Cbln1/GluD2 signaling. Reviews providing a more general overview of Cbln1 and GluD2 have been published elsewhere [12,16,17].
DOI 10.1016/j.conb.2011.01.010
Introduction
Cbln1 and GluD2 — the marriage of an orphan ligand and orphan receptor
The precise apposition of presynaptic and postsynaptic specializations is essential for functional synapses to form and establish precise neuronal circuits. Cell-adhesion molecules, such as neurexin (NRX)/neuroligin (NL) [1], leucine-rich repeat transmembrane neuronal proteins (LRRTMs) [2], synaptic cell-adhesion molecule (SynCAM) [3], and EphB/ephrinB [4], and secreted molecules, such as Wnt-7a [5], fibroblast growth factors [6], thrombospondins [7], and neuronal pentraxins [8] regulate these processes by inducing the differentiation and maturation of presynaptic or postsynaptic structures in vitro, and thus are generally referred to as ‘synaptic organizers’ [9]. Nevertheless, mice lacking a gene encoding an individual synaptic organizer often show little abnormality in their synaptic structures in vivo. Consequently, the requirement of these molecules for synapse maintenance or new synapse formation in the adult CNS remains largely unclear.
GluD2 belongs to the ionotropic glutamate receptor (iGluR) family based on its amino acid sequence [16]. GluD2 has long been referred to as an orphan receptor, because it does not form functional glutamate-gated ion channels when expressed alone or with other iGluRs, in transfected cells. A breakthrough occurred with the unexpected finding that cbln1-null mice show strikingly similar behavioral, physiological, and anatomical phenotypes to those of GluD2-null mice, indicating that Cbln1 and GluD2 might share signaling pathways [10]. Indeed, we found that recombinant Cbln1 specifically binds to the amino-terminal domain (ATD) of GluD2 in vitro [15]. In addition, endogenous Cbln1, which is highly enriched at the synaptic cleft of wild-type PF–Purkinje cell synapses, was not detectable in immunohistochemical assays of the GluD2-null cerebellum. Since the Cbln1 protein levels in wild-type and GluD2-null cerebella are similar, Cbln1 was probably washed off the GluD2-null
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cerebellar sections during the immunostaining procedures. Furthermore, the ATD of GluD2 is indispensable for Cbln1 to induce synapse formation in vitro [15,18] and in vivo [15]. Together, these results indicated that Cbln1 is the missing ligand for the orphan receptor GluD2, and that the interaction between Cbln1 and GluD2 is essential for synapse formation/maintenance in the cerebellum (Figure 1a).
NMDA receptors through a region that contains the ATD of GluN1 [21], leading to the potentiation of NMDA receptor currents. Therefore, a direct ligand–receptor relationship between GluD2 and the extracellular protein Cbln1 is not completely unprecedented. What is unique about GluD2 is that, although it belongs to the iGluR family, it does not require channel activities to achieve its major functions at PF–Purkinje cell synapses [16].
The ATD of the iGluRs is the region most distal from the membrane, and thus a strategically suitable site for interactions with extracellular proteins (Figure 1b). Indeed, the neuronal pentraxins NP1 and Narp, which are secreted from presynaptic and postsynaptic sites, bind to the ATD of the GluA4 subunit of the AMPA receptor and cause these receptors to cluster at the postsynaptic site [19]. Similarly, the ATD of GluA2 regulates dendritic spine formation by interacting with the adhesion molecule N-cadherin [20]. Moreover, EphrinB binding to the EphB receptor enables an interaction between EphB and
NRX — a common presynaptic target for NL, LRRTM, and Cbln1 Human embryonic kidney 293 (HEK293) cells expressing NL or LRRTM form artificial synapses with neurons by interacting with certain isoforms of NRXs at presynaptic sites. For example, LRRTM2 specifically binds NRXb(S4 ), which lacks a splice-site 4 (S4) insert, whereas NL1( ), which lacks splice sites A and B, binds to both NRXb(S4 ) and NRXb(S4+) (Figure 2) [22,23]. Interestingly, adding Cbln1 to the culture medium significantly inhibits synapse formation between cbln1-null
Figure 1
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The amino-terminal domains (ATDs) of iGluRs serve as the site for interactions with extracellular proteins. (a) The NRX/Cbln1/GluD2 tripartite complex is located at the synaptic cleft and functions as a bidirectional synaptic organizer. Cbln1 released from the parallel fiber (PF) accumulates synaptic vesicles (SVs) by binding to neurexin containing splice-site 4 [NRX(S4+)] and induces the clustering of AMPA receptors (GluAs), PSD93/95, homer, and shank, by binding to the postsynaptic glutamate receptor d2 (GluD2). (b) Signaling caused by binding to the ATDs of iGluRs. The neuronal pentraxins NP1 and Narp, secreted from presynaptic and postsynaptic sites, cause clustering of the GluA1 and GluA4 subunits of the AMPA receptor (left). The ATD of GluA2 regulates dendritic spine formation by interacting with the adhesion molecule N-cadherin (middle). The EphrinB–EphB interaction enhances NMDA receptor activities by binding to a region that includes the ATD of the GluN1 subunit (right). Current Opinion in Neurobiology 2011, 21:215–220
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Figure 2
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A model for synaptic signaling by the tripartite complex NRX/Cbln/ GluD. Cbln1 and Cbln2 bind to presynaptic NRXa and NRXb containing splice-site 4 (S4+) and postsynaptic GluD1 and GluD2. While LRRTMs and NLs bind to the NRXs in a Ca2+-dependent manner, the NRX/Cbln/ GluD complex is resistant to low Ca2+ concentration. Unlike LRRTMs, which bind to NRXs(S4 ) and mediate excitatory synaptogenesis, Cbln/GluD specifically binds to NRXs(S4+) and regulates excitatory and inhibitory synaptogenesis, depending on the type of neuron expressing the Cbln protein.
granule cells and HEK293 cells expressing NL1( ), but not those expressing LRRTM2 [24]. Thus, we hypothesized that Cbln1 may interact with NRXb(S4+) expressed at presynaptic sites in granule cells. Indeed, we found that Cbln1 specifically binds to HEK293 cells expressing three subtypes of NRXb [NRX1b(S4+), NRX2b(S4+), and NRX3b(S4+)] and one NRX1a [NRX1a(S4+)], all of which contain the S4 insert, but not to subtypes lacking the S4 insert (Figure 2) [24]. Similarly, using a chemical crosslinking technique to isolate interaction partners of GluD2, Mishina and colleagues identified NRXb(S4+) and Cbln1 as the link to the presynaptic membrane [14]. Immunocytochemical analyses also showed that the ectodomain of NRX1b(S4+), but not that of NRX1b(S4 ), specifically binds to HEK293 cells expressing GluD2 only when Cbln1 is added to the culture medium [24]. These findings indicated that Cbln1 forms a tripartite complex, NRX/Cbln1/GluD2, at the synaptic site. Cbln1-coated beads accumulate synaptic vesicles in cbln1-null granule cell axons [15], and adding the ectodomain of NRX1b(S4+), but not of NRX1b(S4 ), to the culture medium significantly inhibits Cbln1’s presynaptic organizing function [24]. Similarly, Cbln1’s synaptogenic activity in primary cultures and in vivo is inhibited by the ectodomain of NRX1b(S4+) [14]. On the other www.sciencedirect.com
hand, beads coated with NRX1b(S4+), but not with NRX1b(S4 ), cause the clustering of GluD2 and its intracellular-interacting protein shank2 in HEK293 cells or cbln1-null Purkinje cells only when recombinant Cbln1 is added to the culture medium [24]. Therefore, Cbln1 serves as a bidirectional synaptic organizer by interacting with its presynaptic receptor NRX and postsynaptic receptor GluD2.
The NRX/Cbln1/GluD2 complex is a unique synaptic organizer in several ways. First, although the alternative splicing of NRXs and NLs is implicated in specifying synaptic connections [25], major splice-site 4 has only a limited effect on the NRX/NL binding affinities [26]. Instead, LRRTMs bind specifically to NRXb(S4 ) [23,27,28], and now Cbln1 has been found to bind specifically to NRXb(S4+) [14,24]. Furthermore, NRX1a(S4+) binds to Cbln1 [24], whereas it does not significantly bind to any NLs or LRRTMs. These findings suggest that the major function of the alternative splicing at the S4 site may not be to regulate the interaction between NRXs and NLs, but rather to modulate interactions with other binding partners, such as LRRTMs and Cbln1. Second, although the binding of NLs and LRRTMs to NRXs requires extracellular Ca2+ [23,28], the NRX/ Cbln1/GluD2 complex is insensitive to the extracellular Ca2+ concentration [24]. Indeed, Cbln1 still binds to a mutant NRX1b(S4+) [24] in which the Ca2+-binding sites are disrupted [29,30]. Thus, Cbln1 probably binds the region involving the S4 site of NRXs in a manner distinct from NLs or LRRTMs (Figure 2). Although various cell-adhesion molecules, such as cadherins, NRXs/NLGs, and NRXs/LRRTMs, require extracellular Ca2+, synaptic adhesion itself is reported to be insensitive to Ca2+ [31]. Therefore, the Ca2+-independent binding of the NRX/Cbln1/GluD2 complex is consistent with its function as synaptic glue, connecting presynaptic and postsynaptic elements. A third unique feature of the NRX/Cbln1/GluD2 signaling is that the secreted Cbln1 functions by being sandwiched between presynaptic NRX and postsynaptic GluD2. In contrast, other known soluble synaptic organizers in the CNS act at either the presynaptic or postsynaptic site, depending on the location of their receptors [32]. Spines on the distal dendrites of Purkinje cells are believed to be autonomously generated in the absence of presynaptic sites to make as many synapses as possible (100 000 synapses per Purkinje cell) with the numerous orthogonally oriented PFs [33]. We speculate that synaptogenesis by the sandwich-type signaling elicited by the NRX/Cbln1/GluD2 complex may be suitable for this type of synapse. Cbln1 may be broadly located along PF axons by binding to NRXs before synapses are made, and Current Opinion in Neurobiology 2011, 21:215–220
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Cbln1’s encounter with the preexisting GluD2 on Purkinje cell spines may induce clustering and stabilizing the Cbln1 for rapid synaptogenesis. The finding that endogenous Cbln1 located along PFs is not stable without GluD2 [34] is consistent with this scenario.
Activity-dependent regulation of NRX/Cbln1/ GluD2 signaling Cbln1 and GluD2 are differentially regulated at the transcriptional [35] and post-translational [36] levels (Figure 3). Furthermore, the splicing of S4 of NRXs is also regulated during development [37] and by ischemia [38]. A crystallographic study recently revealed that the ligand-binding domain of GluD2 closes upon D-Ser binding [39]. High concentrations of D-Ser are present in cerebellar tissues during the early postnatal period [40] and D-Ser is shown to be released from astrocytes in an activity-dependent manner [41]. Thus, the activity-dependent binding of D-Ser to the ligand-binding site of GluD2 might also modify the synaptogenic activity of the NRX/Cbln1/GluD2 complex in the cerebellum during development, by allosterically affecting the ATD conformation (Figure 3). Such activity-dependent regulation of Figure 3
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Potential activity-dependent regulation of NRX/Cbln1/GluD2 signaling. The expression of cbln1 mRNA is downregulated in presynaptic granule cells when the neuronal activity is increased for hours. The splicing of NRX S4 is also regulated during development and by neuronal activity. In addition, a sustained increase in neuronal activity causes the internalization of GluD2 from the postsynaptic site of Purkinje cells. Furthermore, D-Ser, which is released from Bergmann glia after the burst stimulation of PFs in immature cerebellum, may affect the synaptogenic activity of the NRX/Cbln1/GluD2 complex. Current Opinion in Neurobiology 2011, 21:215–220
each component of the NRX/Cbln1/GluD2 complex might lead to switching between different synaptic organizers in the cerebellum.
Synapses outside the cerebellum Cbln1 mRNA is also enriched in a subset of neurons in various brain regions, including the mitral layer of the olfactory bulb, the entorhinal cortex, and the thalamic parafascicular nucleus [42]. In cbln1-null mice, the spine density of the medial spiny neurons in the striatum, which receive inputs from the Cbln1-positive thalamic parafascicular nucleus, is markedly increased, suggesting that Cbln1 determines the dendritic structure of striatal neurons with effects distinct from those seen in the cerebellum [43]. Although GluD2 is not expressed in these brain regions, its family member GluD1, which also binds to HA-Cbln1 [15], is expressed in them [44]. Interestingly, although Cbln1-coated beads can induce the accumulation of presynaptic terminals of hippocampal and cortical neurons, they fail to accumulate AMPA receptors in hippocampal neurons [24]. Therefore, GluD1 may mediate postsynaptic effects distinct from those regulated by GluD2. Interestingly, human GRID1 gene is suggested to be associated with schizophrenia [45,46]. Of the Cbln family, Cbln2 and Cbln4 are also expressed in various brain regions [42]. Interestingly, Cbln1 and Cbln2 but not Cbln4 can bind to NRX1b(S4+) and induce the hemisynaptic differentiation of cerebellar, hippocampal, and cortical neurons in vitro [24]. Since the predicted amino acid sequences of Cbln1, Cbln2, and Cbln4 are very similar to each other (87–91% identity) [12], such differential effects may help determine the region responsible for the binding to NRXs. Since Cbln4 is coexpressed with Cbln1 or Cbln2 in most brain regions [42], Cbln4 may modulate the synaptogenic activities of Cbln1 and Cbln2 by forming a heteromeric complex.
Conclusions The unique NRX/Cbln1/GluD2 tripartite complex is necessary for both synapse formation and the maintenance of mature PF–Purkinje-cell synapses in vitro and in vivo, by providing bidirectional signals to presynaptic and postsynaptic sites (Figure 1a). Several key questions remain to be addressed in future studies. First, although Cbln1 causes the clustering of postsynaptic molecules via the C-terminus of GluD2 (Figure 1a), whether intracellular signaling is dynamically regulated by the NRX–Cbln1–GluD2 interaction remains unclear. Of particular interest is whether and how AMPA receptor trafficking, which mediates the synaptic plasticity at PF–Purkinje cell synapses, is regulated by GluD2’s binding to D-Ser and Cbln1. Second, the signaling mediated by other family members, such as Cbln2, Cbln4, and GluD1, remains to be clarified in vitro and in vivo. The classical complement C1q has been shown to regulate the selective elimination of inappropriate www.sciencedirect.com
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synaptic connections during development [47]. Members of another C1q family, C1ql, are expressed and secreted in brain regions where Cbln proteins are not expressed [48]. Recently, addition of recombinant C1ql proteins was shown to cause a significant decrease in synapse density in hippocampal neurons in vitro [49]. An understanding of Cbln1’s functions will provide new insights into the roles of other C1q family proteins that regulate normal and abnormal brain functions.
Acknowledgements This review describes studies to which many current and past laboratory members have contributed. This work was supported by Grants-in-Aid from MEXT, the Takeda Science Foundation, and the Core Research for Evolutional Science and Technology of the Japan Science and Technology Agency.
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47. Stevens B, Allen NJ, Vazquez LE, Howell GR, Christopherson KS, Nouri N, Micheva KD, Mehalow AK, Huberman AD, Stafford B et al.: The classical complement cascade mediates CNS synapse elimination. Cell 2007, 131:1164-1178. This study showed that the classical complement C1q controls synapse elimination during development, indicating that the C1q family may generally regulate various aspects of synapses in the CNS. 48. Iijima T, Miura E, Watanabe M, Yuzaki M: Distinct expression of C1q-like family mRNAs in mouse brain and biochemical characterization of their encoded proteins. Eur J Neurosci 2010, 31:1606-1615. 49. Bolliger MF, Martinelli DC, Sudhof TC: The cell-adhesion G protein-coupled receptor BAI3 is a high-affinity receptor for C1q-like proteins. Proc Natl Acad Sci U S A 2011, 108:2534-2539.
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Cbln1 and its family proteins in synapse formation and maintenance Michisuke Yuzaki Cbln1 is a newly identified synaptic organizer belonging to the C1q family. Unlike other synaptic organizers, a deficiency in Cbln1 is sufficient to cause a severe reduction in the number of synapses between cerebellar Purkinje cells and parallel fibers (PFs). Furthermore, Cbln1 can rapidly induce synaptogenesis and is necessary for maintaining normal synapses in the mature cerebellum in vivo. Cbln1 was recently identified as the missing ligand for the orphan glutamate receptor d2 (GluD2), which is expressed in Purkinje cells. Furthermore, Cbln1 released from PFs binds to neurexin (NRX) expressed on the presynaptic PFs and GluD2 at the postsynaptic site. The NRX/Cbln1/GluD2 tripartite complex is resistant to low extracellular Ca2+ levels and serves as a unique bidirectional synaptic organizer. Address Department of Neurophysiology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan Corresponding author: Yuzaki, Michisuke ([email protected])
Cbln1 is a newly identified synaptic organizer released from granule cells in the cerebellum [10]. Cbln1 belongs to the C1q family [11], most of which are secreted (e.g. the complement C1q and adipokine adiponectin), and play diverse roles in intercellular communication [12]. Unlike other synaptic organizers, a deficiency in Cbln1 alone is sufficient to cause a severe reduction in the number of synapses between Purkinje cells and parallel fibers (PFs; axons of granule cells), which results in cerebellar ataxia in mice [10]. A single injection of recombinant Cbln1 into the subarachnoid supracerebellar space rapidly restores PF–Purkinje cell synapses and rescues the ataxia in adult cbln1-null mice within a day, but the effect is transient, and the synapses are lost when the injected Cbln1 is degraded [13]. Similarly, ablation of Cbln1 in the adult cerebellum resulted in a severe reduction in the number of PF– Purkinje cell synapses [14]. Therefore, Cbln1 is a unique synaptic organizer that can rapidly induce synaptogenesis and is necessary for maintaining normal synapses in the mature brain in vivo.
Current Opinion in Neurobiology 2011, 21:215–220 This review comes from a themed issue on Synaptic function and regulation Edited by Yukiko Goda and Bernardo Sabatini Available online 20th February 2011 0959-4388/$ – see front matter # 2011 Elsevier Ltd. All rights reserved.
Recently, two receptors for Cbln1 were identified. One, NRX, is located at the presynaptic site of PF–Purkinje cell synapses; the other, glutamate receptor d2 (GluD2), is postsynaptic [14,15]. In this review, I will summarize the most recent findings regarding NRX/Cbln1/GluD2 signaling. Reviews providing a more general overview of Cbln1 and GluD2 have been published elsewhere [12,16,17].
DOI 10.1016/j.conb.2011.01.010
Introduction
Cbln1 and GluD2 — the marriage of an orphan ligand and orphan receptor
The precise apposition of presynaptic and postsynaptic specializations is essential for functional synapses to form and establish precise neuronal circuits. Cell-adhesion molecules, such as neurexin (NRX)/neuroligin (NL) [1], leucine-rich repeat transmembrane neuronal proteins (LRRTMs) [2], synaptic cell-adhesion molecule (SynCAM) [3], and EphB/ephrinB [4], and secreted molecules, such as Wnt-7a [5], fibroblast growth factors [6], thrombospondins [7], and neuronal pentraxins [8] regulate these processes by inducing the differentiation and maturation of presynaptic or postsynaptic structures in vitro, and thus are generally referred to as ‘synaptic organizers’ [9]. Nevertheless, mice lacking a gene encoding an individual synaptic organizer often show little abnormality in their synaptic structures in vivo. Consequently, the requirement of these molecules for synapse maintenance or new synapse formation in the adult CNS remains largely unclear.
GluD2 belongs to the ionotropic glutamate receptor (iGluR) family based on its amino acid sequence [16]. GluD2 has long been referred to as an orphan receptor, because it does not form functional glutamate-gated ion channels when expressed alone or with other iGluRs, in transfected cells. A breakthrough occurred with the unexpected finding that cbln1-null mice show strikingly similar behavioral, physiological, and anatomical phenotypes to those of GluD2-null mice, indicating that Cbln1 and GluD2 might share signaling pathways [10]. Indeed, we found that recombinant Cbln1 specifically binds to the amino-terminal domain (ATD) of GluD2 in vitro [15]. In addition, endogenous Cbln1, which is highly enriched at the synaptic cleft of wild-type PF–Purkinje cell synapses, was not detectable in immunohistochemical assays of the GluD2-null cerebellum. Since the Cbln1 protein levels in wild-type and GluD2-null cerebella are similar, Cbln1 was probably washed off the GluD2-null
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cerebellar sections during the immunostaining procedures. Furthermore, the ATD of GluD2 is indispensable for Cbln1 to induce synapse formation in vitro [15,18] and in vivo [15]. Together, these results indicated that Cbln1 is the missing ligand for the orphan receptor GluD2, and that the interaction between Cbln1 and GluD2 is essential for synapse formation/maintenance in the cerebellum (Figure 1a).
NMDA receptors through a region that contains the ATD of GluN1 [21], leading to the potentiation of NMDA receptor currents. Therefore, a direct ligand–receptor relationship between GluD2 and the extracellular protein Cbln1 is not completely unprecedented. What is unique about GluD2 is that, although it belongs to the iGluR family, it does not require channel activities to achieve its major functions at PF–Purkinje cell synapses [16].
The ATD of the iGluRs is the region most distal from the membrane, and thus a strategically suitable site for interactions with extracellular proteins (Figure 1b). Indeed, the neuronal pentraxins NP1 and Narp, which are secreted from presynaptic and postsynaptic sites, bind to the ATD of the GluA4 subunit of the AMPA receptor and cause these receptors to cluster at the postsynaptic site [19]. Similarly, the ATD of GluA2 regulates dendritic spine formation by interacting with the adhesion molecule N-cadherin [20]. Moreover, EphrinB binding to the EphB receptor enables an interaction between EphB and
NRX — a common presynaptic target for NL, LRRTM, and Cbln1 Human embryonic kidney 293 (HEK293) cells expressing NL or LRRTM form artificial synapses with neurons by interacting with certain isoforms of NRXs at presynaptic sites. For example, LRRTM2 specifically binds NRXb(S4 ), which lacks a splice-site 4 (S4) insert, whereas NL1( ), which lacks splice sites A and B, binds to both NRXb(S4 ) and NRXb(S4+) (Figure 2) [22,23]. Interestingly, adding Cbln1 to the culture medium significantly inhibits synapse formation between cbln1-null
Figure 1
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The amino-terminal domains (ATDs) of iGluRs serve as the site for interactions with extracellular proteins. (a) The NRX/Cbln1/GluD2 tripartite complex is located at the synaptic cleft and functions as a bidirectional synaptic organizer. Cbln1 released from the parallel fiber (PF) accumulates synaptic vesicles (SVs) by binding to neurexin containing splice-site 4 [NRX(S4+)] and induces the clustering of AMPA receptors (GluAs), PSD93/95, homer, and shank, by binding to the postsynaptic glutamate receptor d2 (GluD2). (b) Signaling caused by binding to the ATDs of iGluRs. The neuronal pentraxins NP1 and Narp, secreted from presynaptic and postsynaptic sites, cause clustering of the GluA1 and GluA4 subunits of the AMPA receptor (left). The ATD of GluA2 regulates dendritic spine formation by interacting with the adhesion molecule N-cadherin (middle). The EphrinB–EphB interaction enhances NMDA receptor activities by binding to a region that includes the ATD of the GluN1 subunit (right). Current Opinion in Neurobiology 2011, 21:215–220
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Figure 2
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A model for synaptic signaling by the tripartite complex NRX/Cbln/ GluD. Cbln1 and Cbln2 bind to presynaptic NRXa and NRXb containing splice-site 4 (S4+) and postsynaptic GluD1 and GluD2. While LRRTMs and NLs bind to the NRXs in a Ca2+-dependent manner, the NRX/Cbln/ GluD complex is resistant to low Ca2+ concentration. Unlike LRRTMs, which bind to NRXs(S4 ) and mediate excitatory synaptogenesis, Cbln/GluD specifically binds to NRXs(S4+) and regulates excitatory and inhibitory synaptogenesis, depending on the type of neuron expressing the Cbln protein.
granule cells and HEK293 cells expressing NL1( ), but not those expressing LRRTM2 [24]. Thus, we hypothesized that Cbln1 may interact with NRXb(S4+) expressed at presynaptic sites in granule cells. Indeed, we found that Cbln1 specifically binds to HEK293 cells expressing three subtypes of NRXb [NRX1b(S4+), NRX2b(S4+), and NRX3b(S4+)] and one NRX1a [NRX1a(S4+)], all of which contain the S4 insert, but not to subtypes lacking the S4 insert (Figure 2) [24]. Similarly, using a chemical crosslinking technique to isolate interaction partners of GluD2, Mishina and colleagues identified NRXb(S4+) and Cbln1 as the link to the presynaptic membrane [14]. Immunocytochemical analyses also showed that the ectodomain of NRX1b(S4+), but not that of NRX1b(S4 ), specifically binds to HEK293 cells expressing GluD2 only when Cbln1 is added to the culture medium [24]. These findings indicated that Cbln1 forms a tripartite complex, NRX/Cbln1/GluD2, at the synaptic site. Cbln1-coated beads accumulate synaptic vesicles in cbln1-null granule cell axons [15], and adding the ectodomain of NRX1b(S4+), but not of NRX1b(S4 ), to the culture medium significantly inhibits Cbln1’s presynaptic organizing function [24]. Similarly, Cbln1’s synaptogenic activity in primary cultures and in vivo is inhibited by the ectodomain of NRX1b(S4+) [14]. On the other www.sciencedirect.com
hand, beads coated with NRX1b(S4+), but not with NRX1b(S4 ), cause the clustering of GluD2 and its intracellular-interacting protein shank2 in HEK293 cells or cbln1-null Purkinje cells only when recombinant Cbln1 is added to the culture medium [24]. Therefore, Cbln1 serves as a bidirectional synaptic organizer by interacting with its presynaptic receptor NRX and postsynaptic receptor GluD2.
The NRX/Cbln1/GluD2 complex is a unique synaptic organizer in several ways. First, although the alternative splicing of NRXs and NLs is implicated in specifying synaptic connections [25], major splice-site 4 has only a limited effect on the NRX/NL binding affinities [26]. Instead, LRRTMs bind specifically to NRXb(S4 ) [23,27,28], and now Cbln1 has been found to bind specifically to NRXb(S4+) [14,24]. Furthermore, NRX1a(S4+) binds to Cbln1 [24], whereas it does not significantly bind to any NLs or LRRTMs. These findings suggest that the major function of the alternative splicing at the S4 site may not be to regulate the interaction between NRXs and NLs, but rather to modulate interactions with other binding partners, such as LRRTMs and Cbln1. Second, although the binding of NLs and LRRTMs to NRXs requires extracellular Ca2+ [23,28], the NRX/ Cbln1/GluD2 complex is insensitive to the extracellular Ca2+ concentration [24]. Indeed, Cbln1 still binds to a mutant NRX1b(S4+) [24] in which the Ca2+-binding sites are disrupted [29,30]. Thus, Cbln1 probably binds the region involving the S4 site of NRXs in a manner distinct from NLs or LRRTMs (Figure 2). Although various cell-adhesion molecules, such as cadherins, NRXs/NLGs, and NRXs/LRRTMs, require extracellular Ca2+, synaptic adhesion itself is reported to be insensitive to Ca2+ [31]. Therefore, the Ca2+-independent binding of the NRX/Cbln1/GluD2 complex is consistent with its function as synaptic glue, connecting presynaptic and postsynaptic elements. A third unique feature of the NRX/Cbln1/GluD2 signaling is that the secreted Cbln1 functions by being sandwiched between presynaptic NRX and postsynaptic GluD2. In contrast, other known soluble synaptic organizers in the CNS act at either the presynaptic or postsynaptic site, depending on the location of their receptors [32]. Spines on the distal dendrites of Purkinje cells are believed to be autonomously generated in the absence of presynaptic sites to make as many synapses as possible (100 000 synapses per Purkinje cell) with the numerous orthogonally oriented PFs [33]. We speculate that synaptogenesis by the sandwich-type signaling elicited by the NRX/Cbln1/GluD2 complex may be suitable for this type of synapse. Cbln1 may be broadly located along PF axons by binding to NRXs before synapses are made, and Current Opinion in Neurobiology 2011, 21:215–220
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Cbln1’s encounter with the preexisting GluD2 on Purkinje cell spines may induce clustering and stabilizing the Cbln1 for rapid synaptogenesis. The finding that endogenous Cbln1 located along PFs is not stable without GluD2 [34] is consistent with this scenario.
Activity-dependent regulation of NRX/Cbln1/ GluD2 signaling Cbln1 and GluD2 are differentially regulated at the transcriptional [35] and post-translational [36] levels (Figure 3). Furthermore, the splicing of S4 of NRXs is also regulated during development [37] and by ischemia [38]. A crystallographic study recently revealed that the ligand-binding domain of GluD2 closes upon D-Ser binding [39]. High concentrations of D-Ser are present in cerebellar tissues during the early postnatal period [40] and D-Ser is shown to be released from astrocytes in an activity-dependent manner [41]. Thus, the activity-dependent binding of D-Ser to the ligand-binding site of GluD2 might also modify the synaptogenic activity of the NRX/Cbln1/GluD2 complex in the cerebellum during development, by allosterically affecting the ATD conformation (Figure 3). Such activity-dependent regulation of Figure 3
splicing
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Potential activity-dependent regulation of NRX/Cbln1/GluD2 signaling. The expression of cbln1 mRNA is downregulated in presynaptic granule cells when the neuronal activity is increased for hours. The splicing of NRX S4 is also regulated during development and by neuronal activity. In addition, a sustained increase in neuronal activity causes the internalization of GluD2 from the postsynaptic site of Purkinje cells. Furthermore, D-Ser, which is released from Bergmann glia after the burst stimulation of PFs in immature cerebellum, may affect the synaptogenic activity of the NRX/Cbln1/GluD2 complex. Current Opinion in Neurobiology 2011, 21:215–220
each component of the NRX/Cbln1/GluD2 complex might lead to switching between different synaptic organizers in the cerebellum.
Synapses outside the cerebellum Cbln1 mRNA is also enriched in a subset of neurons in various brain regions, including the mitral layer of the olfactory bulb, the entorhinal cortex, and the thalamic parafascicular nucleus [42]. In cbln1-null mice, the spine density of the medial spiny neurons in the striatum, which receive inputs from the Cbln1-positive thalamic parafascicular nucleus, is markedly increased, suggesting that Cbln1 determines the dendritic structure of striatal neurons with effects distinct from those seen in the cerebellum [43]. Although GluD2 is not expressed in these brain regions, its family member GluD1, which also binds to HA-Cbln1 [15], is expressed in them [44]. Interestingly, although Cbln1-coated beads can induce the accumulation of presynaptic terminals of hippocampal and cortical neurons, they fail to accumulate AMPA receptors in hippocampal neurons [24]. Therefore, GluD1 may mediate postsynaptic effects distinct from those regulated by GluD2. Interestingly, human GRID1 gene is suggested to be associated with schizophrenia [45,46]. Of the Cbln family, Cbln2 and Cbln4 are also expressed in various brain regions [42]. Interestingly, Cbln1 and Cbln2 but not Cbln4 can bind to NRX1b(S4+) and induce the hemisynaptic differentiation of cerebellar, hippocampal, and cortical neurons in vitro [24]. Since the predicted amino acid sequences of Cbln1, Cbln2, and Cbln4 are very similar to each other (87–91% identity) [12], such differential effects may help determine the region responsible for the binding to NRXs. Since Cbln4 is coexpressed with Cbln1 or Cbln2 in most brain regions [42], Cbln4 may modulate the synaptogenic activities of Cbln1 and Cbln2 by forming a heteromeric complex.
Conclusions The unique NRX/Cbln1/GluD2 tripartite complex is necessary for both synapse formation and the maintenance of mature PF–Purkinje-cell synapses in vitro and in vivo, by providing bidirectional signals to presynaptic and postsynaptic sites (Figure 1a). Several key questions remain to be addressed in future studies. First, although Cbln1 causes the clustering of postsynaptic molecules via the C-terminus of GluD2 (Figure 1a), whether intracellular signaling is dynamically regulated by the NRX–Cbln1–GluD2 interaction remains unclear. Of particular interest is whether and how AMPA receptor trafficking, which mediates the synaptic plasticity at PF–Purkinje cell synapses, is regulated by GluD2’s binding to D-Ser and Cbln1. Second, the signaling mediated by other family members, such as Cbln2, Cbln4, and GluD1, remains to be clarified in vitro and in vivo. The classical complement C1q has been shown to regulate the selective elimination of inappropriate www.sciencedirect.com
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synaptic connections during development [47]. Members of another C1q family, C1ql, are expressed and secreted in brain regions where Cbln proteins are not expressed [48]. Recently, addition of recombinant C1ql proteins was shown to cause a significant decrease in synapse density in hippocampal neurons in vitro [49]. An understanding of Cbln1’s functions will provide new insights into the roles of other C1q family proteins that regulate normal and abnormal brain functions.
Acknowledgements This review describes studies to which many current and past laboratory members have contributed. This work was supported by Grants-in-Aid from MEXT, the Takeda Science Foundation, and the Core Research for Evolutional Science and Technology of the Japan Science and Technology Agency.
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