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 present study tested the effects of EUK-134, a synthetic superoxide dismutase͞catalase mimetic, on several indices of oxidative stress and neuropathology produced in the rat limbic system as a result of seizure activity elicited by systemic kainic acid (KA) administration. Pretreatment of rats with EUK-134 did not modify the latency for or duration of KA-induced seizure activity. It did produce a highly significant reduction in increased protein nitration, activator protein-1-and NF-B-binding activity, and spectrin proteolysis as well as in neuronal damage resulting from seizure activity in limbic structures. These results support the hypothesis that kainate-induced excitotoxicity is caused, at least in part, by the action of reactive oxygen species. Furthermore, they suggest that synthetic superoxide dismutase͞catalase mimetics such as EUK-134 might be used to prevent excitotoxic neuronal injury.Various mechanisms have been proposed to account for the pathological manifestations observed after systemic administration of the excitotoxin kainic acid (KA). Because drugs blocking seizure activity prevent most of the neuronal damage resulting from KA injection (1, 2), it is clear that the pathology is not a direct consequence of the activation of KA or ␣-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors, but rather is the result of events triggered by seizure activity. Excessive production of oxygen-free radicals and other radical species often has been proposed to play an important role in neuronal pathology resulting from excitotoxic insults (3-8). It generally is admitted that KA administration results in the activation of N-methyl-D-aspartate (NMDA) receptors in vulnerable neuronal populations (9, 10), an event that has been shown to cause the formation of superoxide radicals (11,12). Moreover, we reported previously that levels of lipid peroxidation and protein oxidation, two parameters of oxidative stress, were increased significantly in hippocampus and piriform cortex at 8 and 16 h after KAinduced seizure activity in adult rats (13). We also showed that the DNA-binding activity of two transcription factors, namely activator protein-1 (AP-1) and NF-B, generally considered to be markers of cellular insults (14, 15), was increased in these structures under these conditions (16). However, a causal relationship between oxidative stress and neuronal damage after systemic kainate injection has not been established unambiguously, because many agents used as antioxidants also interfere with seizure activity. In addition, because excitotoxicity also has been shown to be reduced by caspase inhibitors (17), oxidative stress could be a late consequence of the events triggered by seizure activity and leading to neuronal death.Salen-manganese complexes are low-molecular-weight synthetic compounds that exhibit both superoxide dismutase (SOD) and catalase activities, catalytically eliminating both superoxide and hydrogen peroxide, respectively (18 -20). Compared with proteinaceous antioxidant en...

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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Previous studies have indicated that increased formation of oxygen free radicals is likely to participate in the cascade of events leading to neuronal damage following kainic acid (KA)-induced seizure activity. As reactive oxygen species are involved in signal transduction pathways leading to nuclear factor-KB (NF-KB) activation, we examined the effects of KA treatment on the activation of NF-KB in adult and juvenile rat brain. For comparison, changes in two other transcription factors, activator protein-i (AP-i) and Spi, were also determined. In adult rat piriform cortex and hippocampus, significant induction of NF-KB was observed at 4 h after KA injection, and the maximal increase was reached at 8-16 h posttreatment. NF-KB binding activities returned to control levels by 5 days after injection. NF-KB binding activities were slightly decreased in adult rat cerebellum at 8 and 16 h after KA treatment. In the juvenile rat, no significant changes in NF-KB binding activity were observed in piriform cortex, hippocampus, and cerebellum after KA injection. Changes in AP-i binding activity were qualitatively similar to those observed with NF-KB in adult but not juvenile rat brain, as AP-i was significantly induced in juvenile piriform cortex and hippocampus following KA injection. On the other hand, little or no changes in Spi activity were detected in adult and juvenile rat brain. Our results provide further evidence that oxidative stress participates in neuronal damage resulting from KA-induced seizure activity.

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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.

Both tyrosine phosphorylation and calpain-mediated truncation of ionotropic glutamate receptors are important mechanisms for synaptic plasticity. Previous work from our laboratory has shown that calpain activation results in truncation of the C-terminal domains of several glutamate receptor subunits. To test whether and how tyrosine phosphorylation of glutamate ionotropic receptor subunits modulates calpain susceptibility, synaptic membranes were phosphorylated by Fyn or Src, two members of the Src family tyrosine kinases. Tyrosine phosphorylation of synaptic membranes by Src signi®cantly reduced calpain-mediated truncation of both NR2A and NR2B subunits of NMDA receptors, but not of GluR1 subunits of AMPA receptors. In contrast, phosphorylation with Fyn signi®cantly protected calpain-mediated truncation of GluR1 subunits of AMPA receptors, but enhanced calpain-mediated truncation of NR2A subunits of NMDA receptors. Similar results were observed with NR2A and NR2B C-terminal domain fusion proteins phosphorylated by Fyn or Src before incubation with calpain and calcium. In addition, phosphorylation of NR2A and NR2B C-terminal fusion proteins by Fyn or Src enhanced their binding to spectrin and PSD-95. Thus, tyrosine phosphorylation impairs or facilitates calpainmediated truncation of glutamate receptor subunits, depending on which tyrosine kinase is activated. Such mechanisms could serve to regulate receptor integrity and location, in addition to modulating channel properties.