In 1984, the cerebellum-specific hexadecapeptide “cerebellin” was identified and shown to be concentrated in the synaptosomal compartment (Slemmon et al. 1984). Although “precerebellin” was originally identified as a precursor of cerebellin, the cerebellin peptide is not flanked by the classical dibasic amino acids observed in many neuropeptide precursors (Urade et al. 1991). In addition, precerebellin clearly belongs to the C1q family, whose members, such as C1q, adiponectin (Adipoq), and collagen X, are secreted and are involved in various intercellular functions (Yuzaki 2008). Indeed, full-length, uncleaved precerebellin is secreted from cerebellar granule cells (Bao et al. 2005; Iijima et al. 2007). Therefore, it is now evident that precerebellin is the actual signaling molecule itself, which should be referred to as Cbln1, although the cerebellin peptide may have additional functions.
Cbln1 is predominantly expressed in cerebellar granule cells. Analysis of Cbln1-null mice revealed two essential functions of Cbln1 at synapses formed between parallel fibers (PFs; axons of granule cells) and Purkinje cells in the cerebellum (Hirai et al. 2005). The number of PF-Purkinje cell synapses is markedly reduced, and as many as 80% of the dendritic spines of Purkinje cells remains uninnervated in Cbln1-null mice. In the remaining PF-Purkinje cell synapses in Cbln1−/− mice, the postsynaptic densities (PSDs) are frequently longer than the presynaptic active zones, whereas the length of the active zones completely matches that of PSDs in wild-type mice. In addition, the long-term depression (LTD) of synaptic transmission at PF-Purkinje cell synapses, which is thought to underlie motor coordination and information storage in the cerebellum, is completely abrogated in Cbln1-null cerebellum. These unique phenotypes indicate that Cbln1 functions as a synaptic organizer that regulates matching and the maintenance of pre- and postsynaptic structures and proper synaptic functions at PF-Purkinje cell synapses.
Cbln1 as a C1q Family Protein
Cbln1 belongs to the C1q family, which is characterized by the globular C1q domain (gC1q) at the C-terminus. All C1q family proteins are thought to form trimers via the C-terminal gC1q domain, and this unit trimer is often further organized into a higher-order multimeric complex via various motifs located at the N-terminus. Recent structural analyses of the Cbln1-GluD2 complex have indicated that the gC1q trimer is the minimal unit for interaction with the amino-terminal domain (ATD) of GluD2 (Elegheert et al. 2016). Cbln1 forms a hexamer via the disulfide bond at the N-terminal cysteine-rich region (CRR) (Bao et al. 2005; Iijima et al. 2007), to which one NRX monomer binds (Elegheert, et al. 2016). As one Cbln1 hexamer binds to the GluD2-ATD dimer, overall stoichiometry of NRX monomer/Cbln1 hexamer/GluD2 tetramer tripartite complex is 2:2:1 (Elegheert et al. 2016). Cbln1 with a mutation at the CRR does not form a hexamer and lose synaptogenic activities (Ito-Ishida et al. 2008). Thus, like other C1q family proteins, such as adiponectin and collagen X, the oligomeric status of Cbln1 is crucial for its functions.
The C1q family proteins including Cbln1 are completely absent in plants (Arabidopsis thaliana), yeasts (Saccharomyces cerevisiae, Schizosaccharomyces pombe), nematodes (Caenorhabditis elegans), and insects (Drosophila melanogaster). The Cbln family consists of four members, Cbln1–Cbln4, which share 57–79% amino acid similarity with each other. All Cbln proteins are highly conserved among mammals. Indeed, 96–100% of the amino acid sequences of Cbln1–Cbln4 are identical between mice and humans. In contrast, Cbln3 is absent in frog (Xenopus laevis), fish (Danio rerio), and birds (Gallus gallus). These findings suggest that the Cbln family, especially Cbln3, may have emerged relatively late in the evolution to overcome the requirement for complex synaptic wiring (Yuzaki 2008; Yang et al. 2010).
Cbln1, Cbln2, and Cbln4 are expressed in various brain regions outside the cerebellum (Miura et al. 2006), including different subpopulations of dorsal horn neurons in the mouse spinal cord (Cagle and Honig 2014). Indeed, it has been reported that forebrain-predominant Cbln1-null mice showed impaired fear conditioning and spatial memory (Otsuka et al. 2016). Interestingly, while Cbln1 and Cbln2 bind to NRX and induce the presynaptic differentiation of cerebellar, hippocampal, and cortical neurons in vitro, Cbln4 only weakly binds to NRX showing little synaptogenic activity (Matsuda and Yuzaki 2011). Since Cbln4 is coexpressed with Cbln1 or Cbln2 in many brain regions (Miura et al. 2006), Cbln4 may modulate the synaptogenic activities of Cbln1 and Cbln2 by forming a heteromeric complex. Alternatively, Cbln4 may have distinct binding partners. Indeed, Cbln4, but not Cbln1 or Cbln2, binds to DCC, a netrin receptor in vitro. Thus, heteromeric complex composed of different combinations of Cbln1/2/4 may have variable synaptogenic functions (Wei et al. 2012).
Although GluD2 is predominantly expressed in the cerebellum, its family protein GluD1 is expressed in various brain regions, such as olfactory bulb, hippocampus, cerebral cortex, striatum, and cerebellum (Konno et al. 2014). GluD1 binds to Cbln1 (Matsuda et al. 2010; Uemura et al. 2010) and induces synapse formation in vitro (Ryu et al. 2012; Yasumura et al. 2012). In addition, the density of synapses between molecular layer interneurons and PF is reduced in GluD1-null cerebellum (Konno, et al. 2014), indicating that GluD1 is involved in synapse formation in vivo. In the hippocampus, GluD1 is enriched in the stratum lacunosum-moleculare of the Ammon’s horn and in the middle molecular layer of dentate gyrus (Konno et al. 2014). Interestingly, these regions receive inputs from a subset of neurons in the entorhinal cortex, which expresses Cbln1 or Cbln4 (Miura et al. 2006). These findings suggest that Cbln1/4 derived from these inputs may regulate synaptic functions by forming a tripartite complex NRX/Cbln1/4/GluD1, similar to those achieved by the NRX/Cbln1/ GluD2 complex in the cerebellum.
A New Comer to the World of Synaptic Organizers
In contrast to these conventional synaptic organizers, Cbln1 secreted from PFs is sandwiched between the presynaptic receptor NRXs and the postsynaptic receptor GluD2 (Figs. 1 and 2c). Similarly, glia-derived neurotrophic factor (GDNF) is reported to serve as a synaptic adhesion molecule being sandwiched by its receptor GFRα1 located at both pre- and postsynaptic neurons (Ledda et al. 2007). In addition, leucine-rich glioma inactivated 1 (LGI1) is also secreted from neurons and bind to its pre- and postsynaptic receptors ADAM22 and ADAM23, respectively, to accumulate potassium channels and AMPA receptors at synapses (Fukata et al. 2010). Thus, Cbln1 and these molecules may constitute the third category of synaptic organizers, a sandwich type (Fig. 2c) (Yuzaki 2011).
Interestingly, C1ql2 and C1ql3, which belong to the C1q family, have been recently shown to be released from mossy fibers of the dentate gyrus, bind to the presynaptic NRX3 and determine the synaptic localization of postsynaptic kainate receptors in CA3 pyramidal neurons (Fig. 2c) (Matsuda et al. 2016). Therefore, the transsynaptic tripartite complex composed of NRX, C1q proteins, and the ATD of glutamate receptors may play a general role in bidirectional regulation of synaptic functions in various brain regions.
Cbln1 is one of the synaptic organizers that belong to the C1q family. Unlike other synaptic organizers, a deficiency in Cbln1 causes a severe reduction in the number of synapses between PFs and Purkinje cells in the cerebellum. One GluD2 tetramer binds to two Cbln1 hexamers, each of which bind to one NRX monomers (Elegheert et al. 2016). Such transsynaptic NRX/Cbln1/GluD2 tripartite complex promotes synaptogenesis and regulates the induction of LTD. Functions and signaling mechanisms mediated by other related family members, such as Cbln2, Cbln4, and GluD1, remain to be clarified. Another C1q family protein, C1ql2 and C1ql3 derived from presynaptic sites, recruit kainate receptors to the postsynaptic site (Matsuda et al. 2016). On the other hand, the classical complements C1q and C1ql1 are shown to regulate the elimination of synaptic connections (Stevens et al. 2007; Bolliger et al. 2011; Kakegawa et al. 2015). Thus, characterization of C1q family proteins is expected to provide new insights into the mechanisms by which synapses are formed, matured, and modified in the CNS.
- Elegheert J, Kakegawa W, Clay JE, Shanks NF, Behiels E, Matsuda K, Kohda K, Miura E, Rossmann M, Mitakidis N, Motohashi J, Chang VT, Siebold C, Greger IH, Nakagawa T, Yuzaki M, Aricescu AR. Structural basis for integration of GluD receptors within synaptic organizer complexes. Science. 2016;353:295–9.CrossRefPubMedPubMedCentralGoogle Scholar
- Kakegawa W, Mitakidis N, Miura E, Abe M, Matsuda K, Takeo YH, Kohda K, Motohashi J, Takahashi A, Nagao S, Muramatsu S, Watanabe M, Sakimura K, Aricescu AR, Yuzaki M. Anterograde C1ql1 signaling is required in order to determine and maintain a single-winner climbing fiber in the mouse cerebellum. Neuron. 2015;85:316–29.CrossRefPubMedGoogle Scholar
- Konno K, Matsuda K, Nakamoto C, Uchigashima M, Miyazaki T, Yamasaki M, Sakimura K, Yuzaki M, Watanabe M. Enriched expression of GluD1 in higher brain regions and its involvement in parallel fiber-interneuron synapse formation in the cerebellum. J Neurosci. 2014;34:7412–24.CrossRefPubMedGoogle Scholar
- Matsuda K, Budisantoso T, Mitakidis N, Sugaya Y, Miura E, Kakegawa W, Yamasaki M, Konno K, Uchigashima M, Abe M, Watanabe I, Kano M, Watanabe M, Sakimura K, Aricescu AR, Yuzaki M. Transsynaptic modulation of kainate receptor functions by C1q-like Proteins. Neuron. 2016;90:752–67.Google Scholar
- Otsuka S, Konno K, Abe M, Motohashi J, Kohda K, Sakimura K, Watanabe M, Yuzaki M. Roles of Cbln1 in non-motor functions of mice. J Neurosci. 2016; 36:11801–16.Google Scholar
- Yasumura M, Yoshida T, Lee SJ, Uemura T, Joo JY, Mishina M. Glutamate receptor delta1 induces preferentially inhibitory presynaptic differentiation of cortical neurons by interacting with neurexins through cerebellin precursor protein subtypes. J Neurochem. 2012;121:705–16.CrossRefPubMedGoogle Scholar