Glycosylation of Cblns attenuates their receptor binding.

Cbln1 is the prototype of a family (Cbln1-Cbln4) of secreted glycoproteins and is essential for normal synapse structure and function in cerebellum by bridging presynaptic Nrxn to postsynaptic Grid2. Here we report the effects of glycosylation on the in vitro receptor binding properties of Cblns. Cbln1, 2 and 4 harbor two N-linked glycosylation sites, one at the N-terminus is in a region implicated in Nrxn binding and the second is in the C1q domain, a region involved in Grid2 binding. Mutation (asparagine to glutamine) of the N-terminal site, increased neurexin binding whereas mutation of the C1q site markedly increased Grid2 binding. These mutations did not influence subunit composition of Cbln trimeric complexes (mediated through the C1q domain) nor their assembly into hexamers (mediated by the N-terminal region). Therefore, glycosylation likely masks the receptor binding interfaces of Cblns. As Cbln4 has undetectable Grid2 binding in vitro we assessed whether transgenic expression of wild type Cbln4 or its glycosylation mutants rescued the Cbln1-null phenotype in vivo. Cbln4 partially rescued and both glycosylation mutants completely rescued ataxia in cbln1-null mice. Thus Cbln4 has intrinsic Grid2 binding that is attenuated by glycosylation, and glycosylation mutants exhibit gain of function in vivo.

The Cbln family of proteins interact with multiple signaling pathways.

Cerebellin precursor protein (Cbln1) is essential for synapse integrity in cerebellum through assembly into complexes that bridge pre-synaptic ß-neurexins (Nrxn) to post-synaptic 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, (deleted in colorectal cancer (DCC) 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 Cbln 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.

Comparison of Cbln1 and Cbln2 functions using transgenic and knockout mice.

Cerebellin precursor protein 1 (Cbln1) is the prototype of a family of secreted neuronal glycoproteins (Cbln1-4) and its genetic elimination results in synaptic alterations in cerebellum (CB) and striatum. In CB, Cbln1 acts as a bi-functional ligand bridging pre-synaptic ß-neurexins on granule cells to post-synaptic Grid2 on Purkinje neurons. Although much is known concerning the action of Cbln1, little is known of the function of its other family members. Here, we show that Cbln1 and Cbln2 have similar binding activities to ß-neurexins and Grid2 and the targeted ectopic expression of Cbln2 to Purkinje cells in transgenic mice rescues the cerebellar deficits in Cbln1-null animals: suggesting that the two proteins have redundant function mediated by their common receptor binding properties. Cbln1 and Cbln2 are also co-expressed in the endolysosomal compartment of the thalamic neurons responsible for the synaptic alterations in striatum of Cbln1-null mice. Therefore, to determine whether the two family members have similar functions, we generated Cbln2-null mice. Cbln2-null mice do not show the synaptic alterations evident in striatum of Cbln1-null mice. Thus, Cbln2 can exhibit functional redundancy with Cbln1 in CB but it does not have the same properties as Cbln1 in thalamic neurons, implying one or both utilize different receptors/mechanisms in this brain region.

Mapping of Cbln1-like immunoreactivity in adult and developing mouse brain and its localization to the endolysosomal compartment of neurons.

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 interaction-dependent secretion of Cbln3.

Cbln1 and the orphan glutamate receptor GluRdelta2 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.

The carboxypeptidase-like substrate-binding site in Nna1 is essential for the rescue of the Purkinje cell degeneration (pcd) phenotype.

The Purkinje cell degeneration (pcd) phenotype is characterized by adult onset neurodegeneration resulting from mutations in Nna1, a gene encoding an intracellular protein with a putative metallocarboxypeptidase domain. As Nna1 is also induced in axotomized motor neurons, the elucidation of its function can shed light on previously unsuspected mechanisms common to degenerative and regenerative responses. Structural modeling revealed that Nna1 and three related gene products constitute a new subfamily of metallocarboxypeptidases with a distinctive substrate-binding site. To test whether the metallocarboxypeptidase domain is functionally essential, transgenic mice were generated that expressed Nna1 or a substrate-binding site mutant of Nna1 selectively in Purkinje cells using the L7/pcp2 promoter. When bred onto a homozygous pcd(3J) background, wild type but not mutant Nna1 rescued ataxic behavior and Purkinje cell loss. Therefore, loss of Nna1 in Purkinje cells leads directly to their degeneration and Nna1's carboxypeptidase domain is essential for survival of these neurons.

Cbln1 is essential for synaptic integrity and plasticity in the cerebellum.

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.