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.

The hexadecapeptide cerebellin is present in the brains of many vertebrate species and is derived from a larger protein, Cbln1 (cerebellin 1 precursor protein). Although cerebellin has features of a neuropeptide, Cbln1 belongs to the C1q/ tumor necrosis factor superfamily of secreted proteins, suggesting that it is the biologically active molecule and the proteolytic events that generate cerebellin serve another function. Therefore, we assessed whether Cbln1 undergoes proteolytic processing and determined what consequences the cleavage events necessary to produce cerebellin have on the structure of Cbln1. Substantial degradation of Cbln1 was evident in the synaptic compartment of cerebellum and lysates of cultured cerebellar neurons and cells transfected with Cbln1 expression vectors. However, only uncleaved Cbln1 containing the cerebellin motif was released and assembled into hexameric complexes. Using yeast two hybrid and mammalian expression systems we show that the cleavages required to produce cerebellin influence the subunit stoichiometry of Cbln1 complexes. Cleavage at the Nterminus of the cerebellin sequence in Cbln1 yields trimeric complexes by separating the trimer-mediating C-terminal C1q domain from conserved N-terminal cysteine residues that mediate higher order oligomerization. Cleavage at the Cterminus of the cerebellin motif disrupts the C1q domain and abolishes subunit interactions. Functional implications of these data are discussed.

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.

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, a glycoprotein secreted from granule cells and GluRδ2 in the postsynaptic densities of Purkinje cells are components of an incompletely understood pathway essential for integrity and plasticity of parallel fiber-Purkinje cell synapses. We show that Cbln1 undergoes anterograde transport from granule cells to Purkinje cells and Bergmann glia, and enters the endolysosomal trafficking system, raising the possibility that Cbln1 exerts its activity on or within Purkinje cells and Bergmann glia. Cbln1 is absent in Purkinje cells and Bergmann glia of GluRδ2-null mice, suggesting a mechanistic convergence on Cbln1 trafficking. Ectopic expression of Cbln1 in Purkinje cells of L7-cbln1 transgenic mice reveals Cbln1 undergoes anterograde and retrograde transneuronal trafficking even across synapses that lack GluRδ2, indicating it is not universally essential for Cbln1 transport. The L7-cbln1 transgene also ameliorates the locomotor deficits of cbln1-null mice, indicating the presence and/or release of Cbln1 from the postsynaptic neuron has functional consequences.