Cbln1 is a cerebellum-specific protein of previously unknown function that is structurally related to the C1q and tumor necrosis factor families of proteins. We show that Cbln1 is a glycoprotein secreted from cerebellar granule cells that is essential for three processes in cerebellar Purkinje cells: the matching and maintenance of pre- and postsynaptic elements at parallel fiber-Purkinje cell synapses, the establishment of the proper pattern of climbing fiber-Purkinje cell innervation, and induction of long-term depression at parallel fiber-Purkinje cell synapses. Notably, the phenotype of cbln1-null mice mimics loss-of-function mutations in the orphan glutamate receptor, GluR delta2, a gene selectively expressed in Purkinje neurons. Therefore, Cbln1 secreted from presynaptic granule cells may be a component of a transneuronal signaling pathway that controls synaptic structure and plasticity.

Cbln1 and the orphan glutamate receptor GluR␦2 are pre-and postsynaptic components, respectively, of a novel transneuronal signaling pathway regulating synapse structure and function. We show here that Cbln1 is secreted from cerebellar granule cells in complex with a related protein, Cbln3. However, cbln1-and cbln3-null mice have different phenotypes and cbln1 cbln3 double-null mice have deficits identical to those of cbln1 knockout mice. The basis for these discordant phenotypes is that Cbln1 and Cbln3 reciprocally regulate each other's degradation and secretion such that cbln1-null mice lack both Cbln1 and Cbln3, whereas cbln3-null mice lack Cbln3 but have an approximately sixfold increase in Cbln1. Unlike Cbln1, Cbln3 cannot form homomeric complexes and is secreted only when bound to Cbln1. Structural modeling and mutation analysis reveal that, by constituting a steric clash that is masked upon binding Cbln1 in a "hide-and-run" mechanism of endoplasmic reticulum retention, a single arginine confers the unique properties of Cbln3.

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Cbln1 is a secreted glycoprotein essential for synapse structure and function in cerebellum that is also expressed in extracerebellar structures where its function is unknown. Furthermore, Cbln1 assembles into homomeric complexes and heteromeric complexes with three family members (Cbln2-Cbln4), thereby influencing each other's degradation and secretion. Therefore, to understand its function, it is essential to establish the location of Cbln1 relative to other family members. The localization of Cbln1 in brain was determined using immunohistochemistry and cbln1-lacZ transgenic mice. Cbln1-like immunoreactivity (CLI) was always punctate and localized to the cytoplasm of neurons. The punctate CLI colocalized with cathepsin D, a lysosomal marker, but not with markers of endoplasmic reticulum or Golgi, indicating that Cbln1 is present in neuronal endosomes/lysosomes. This may represent the cellular mechanism underlying the regulated degradation of Cbln1 observed in vivo. Outside the cerebellum, CLI mapped to multiple brain regions that were frequently synaptically interconnected, warranting their analysis in cbln1-null mice. Furthermore, whereas CLI increased dramatically in the cerebellum of cbln3-null mice it was unchanged in extracerebellar neurons. This opens the possibility that other family members that are coexpressed in these areas control Cbln1 levels, potentially by modulating processing in the endolysosomal pathway. During development of cbln1-lacZ mice, beta-galactosidase staining was first observed in proliferating granule cell precursors prior to synaptogenesis and thereafter in maturing and adult granule cells. As cbln3 is only expressed in post-mitotic, post-migratory granule cells, Cbln1 homomeric complexes in precursors and Cbln1-Cbln3 heteromeric complexes in mature granule cells may have distinct functions and turnover.

Cbln1 is essential for synapse integrity in cerebellum through assembly into complexes that bridge presynaptic β-neurexins (Nrxn) to postsynaptic GluRδ2. However, GluRδ2 is largely cerebellum-specific, yet Cbln1 and its little studied family members, Cbln2 and Cbln4, are expressed throughout brain. Therefore, we investigated whether additional proteins mediate Cbln family actions. Whereas Cbln1 and Cbln2 bound to GluRδ2 and Nrxns1–3, Cbln4 bound weakly or not at all, suggesting it has distinct binding partners. In a candidate receptor-screening assay, Cbln4 (but not Cbln1 or Cbln2) bound selectively to the netrin receptor, DCC (deleted in colorectal cancer) in a netrin-displaceable fashion. To determine whether Cbln4 had a netrin-like function, Cbln4-null mice were generated. Cbln4-null mice did not phenocopy netrin-null mice. Cbln1 and Cbln4 were likely co-localized in neurons thought to be responsible for synaptic changes in striatum of Cbln1-null mice. Furthermore, complexes containing Cbln1 and Cbln4 had greatly reduced affinity to DCC but increased affinity to Nrxns, suggesting a functional interaction. However, Cbln4-null mice lacked the striatal synaptic changes seen in Cbln1-null mice. Thus Cbln family members interact with multiple receptors/signaling pathways in a subunit composition-dependent manner and have independent functions with Cbln4 potentially involved in the less-well characterized role of netrin/DCC in adult brain.