No abstract

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.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). With the exception of a few rare familial forms of the disease, the precise molecular mechanisms underlying PD are unknown. Inflammation is a common finding in the PD brain, but due to the limitation of postmortem analysis its relationship to disease progression cannot be established. However, studies using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD have also identified inflammatory responses in the nigrostriatal pathway that precede neuronal degeneration in the SNpc. To assess the pathological relevance of these inflammatory responses and to identify candidate genes that might contribute to neuronal vulnerability, we used quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to measure mRNA levels of 11 cytokine and chemokine encoding genes in the striatum of MPTP-sensitive (C57BL/6J) and MPTP-insensitive (Swiss Webster, SWR) mice following administration of MPTP. The mRNA levels of all 11 genes changed following MPTP treatment, indicating the presence of inflammatory responses in both strains. Furthermore, of the 11 genes examined only 3, interleukin 6 (Il-6), macrophage inflammatory protein 1 alpha/CC chemokine ligand 3 (Mip-1alpha/Ccl3) and macrophage inflammatory protein 1 beta/CC chemokine ligand 4 (Mip-1beta/Ccl4), were differentially regulated between C57BL/6J and SWR mice. In both mouse strains, the level of monocyte chemoattractant protein 1/CC chemokine ligand 2 (Mcp-1/Ccl2) mRNA was the first to increase following MPTP administration, and might represent a key initiating component of the inflammatory response. Using Mcp-1/Ccl2 knockout mice backcrossed onto a C57BL/6J background we found that MPTP-stimulated Mip-1alpha/Ccl3 and Mip-1beta/Ccl4 mRNA expression was significantly lower in the knockout mice; suggesting that Mcp-1/Ccl2 contributes to MPTP-enhanced expression of Mip-1alpha/Ccl3 and Mip-1beta/Ccl4. However, stereological analysis of SNpc neuronal loss in Mcp-1/Ccl2 knockout and wild-type mice showed no differences. These findings suggest that it is the ability of dopaminergic SNpc neurons to survive an inflammatory insult, rather than genetically determined differences in the inflammatory response itself, that underlie the molecular basis of MPTP resistance.

Cbln1 is the prototype of a family of secreted neuronal glycoproteins (Cbln1-4) and its genetic elimination results in synaptic alterations in cerebellum and striatum. In cerebellum, Cbln1 acts as a bi-functional ligand bridging pre-synaptic β-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 β-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 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 cerebellum 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.