Cbln1 Regulates Rapid Formation and Maintenance of Excitatory Synapses in Mature Cerebellar Purkinje Cells In Vitro and In Vivo

Abstract

Although many synapse-organizing molecules have been identified in vitro, their functions in mature neurons in vivo have been mostly unexplored. Cbln1, which belongs to the C1q/tumor necrosis factor superfamily, is the most recently identified protein involved in synapse formation in the mammalian CNS. In the cerebellum, Cbln1 is predominantly produced and secreted from granule cells; cbln1-null mice show ataxia and a severe reduction in the number of synapses between Purkinje cells and parallel fibers (PFs), the axon bundle of granule cells. Here, we show that application of recombinant Cbln1 specifically and reversibly induced PF synapse formation in dissociated cbln1-null Purkinje cells in culture. Cbln1 also rapidly induced electrophysiologically functional and ultrastructurally normal PF synapses in acutely prepared cbln1-null cerebellar slices. Furthermore, a single injection of recombinant Cbln1 rescued severe ataxia in adult cbln1-null mice in vivo by completely, but transiently, restoring PF synapses. Therefore, Cbln1 is a unique synapse organizer that is required not only for the normal development of PF-Purkinje cell synapses but also for their maintenance in the mature cerebellum both in vitro and in vivo. Furthermore, our results indicate that Cbln1 can also rapidly organize new synapses in adult cerebellum, implying its therapeutic potential for cerebellar ataxic disorders.

Activity-Dependent Repression of Cbln1 Expression: Mechanism for Developmental and Homeostatic Regulation of Synapses in the Cerebellum

“…As reported previously (Ito-Ishida et al, 2008), the application of exogenous Cbln1 (2 g/ml, ϳ8.8 nM) had no effects on VGluT1 immunoreactivity on wild-type Purkinje cells (n ϭ 20; p ϭ 0.81). In contrast, the high-K ϩ -induced reduction of VGluT1 immunoreactivity in wild-type Purkinje cells was partially but significantly rescued by the coapplication of exogenous Cbln1 (n ϭ 32; p Ͻ 0.001) (Fig.…”

“…Indeed, unlike in cbln1 Ϫ/Ϫ cerebellum, application of exogenous Cbln1 failed to induce any synaptogenesis in wild-type cerebellum (Ito-Ishida et al, 2008). Nevertheless, changes in neuronal activity reportedly induce considerable structural changes at PF synapses in adult cerebellum in vivo (Black et al, 1990;Cesa et al, 2007).…”

“…To confirm the structural specificity of Cbln1, a mutant Cbln1 (dS-Cbln1), in which cysteine 34 and cysteine 38 were replaced with serines, was also prepared . The concentration of recombinant HA-Cbln1 or HA-dS-Cbln1 in the medium was quantified using an immunoblot analysis with purified 6ϫ histidine-tagged HA-Cbln1 as the standard (Ito-Ishida et al, 2008).…”

“…The original images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD). After subtracting the background level, the mean intensity of presynaptic markers within calbindin-positive dendritic areas distant from the soma by Ͼ20 m was normalized to that of calbindin, as described previously (Ito-Ishida et al, 2008). The number of neurons was regarded as n in the statistical analysis.…”

“…In the cerebellum, Cbln1 is released from granule cells and plays a crucial role in forming excitatory synapses between parallel fibers (PFs; axons of granule cells) and Purkinje cells during development; the number of PFPurkinje cell synapses decreased by 80% in mice with a disrupted cbln1 gene (cbln1 Ϫ/Ϫ ) (Hirai et al, 2005). Interestingly, the single application of recombinant Cbln1 rapidly, but transiently, restored PF synapses in mature cbln1 Ϫ/Ϫ Purkinje cells both in vitro and in vivo (Ito-Ishida et al, 2008). Therefore, Cbln1 is a unique synapse organizer that is required not only for the normal development of PF-Purkinje cell synapses but also for their stabilization in the mature cerebellum.…”