K.M. and T.K. contributed equally to this work.
Cbln1 binds to specific postsynaptic sites at parallel fiber–Purkinje cell synapses in the cerebellum
Version of Record online: 5 FEB 2009
© The Authors (2009). Journal Compilation © Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience
Volume 29, Issue 4, pages 707–717, February 2009
How to Cite
Matsuda, K., Kondo, T., Iijima, T., Matsuda, S., Watanabe, M. and Yuzaki, M. (2009), Cbln1 binds to specific postsynaptic sites at parallel fiber–Purkinje cell synapses in the cerebellum. European Journal of Neuroscience, 29: 707–717. doi: 10.1111/j.1460-9568.2009.06639.x
- Issue online: 16 FEB 2009
- Version of Record online: 5 FEB 2009
- Received 15 November 2008, revised 20 December 2008, accepted 23 December 2008
- granule cell;
- postsynaptic density
Cbln1, which belongs to the C1q/tumor necrosis factor superfamily, is a unique molecule that is not only required for maintaining normal parallel fiber (PF)–Purkinje cell synapses, but is also capable of inducing new PF synapses in adult cerebellum. Although Cbln1 is reportedly released from granule cells, where and how Cbln1 binds in the cerebellum has remained largely unclear, partly because Cbln1 undergoes proteolysis to yield various fragments that are differentially detected by different antibodies. To circumvent this problem, we characterized the Cbln1-binding site using recombinant Cbln1. An immunohistochemical analysis revealed that recombinant Cbln1 preferentially bound to PF–Purkinje cell synapses in primary cultures and acute slice preparations in a saturable and replaceable manner. Specific binding was observed for intact Cbln1 that had formed a hexamer, but not for the N-terminal or C-terminal fragments of Cbln1 fused to other proteins. Similarly, mutant Cbln1 that had formed a trimer did not bind to the Purkinje cells. Immunoreactivity for the recombinant Cbln1 was observed in weaver cerebellum (which lacks granule cells) but was absent in pcd cerebellum (which lacks Purkinje cells), suggesting that the binding site was located on the postsynaptic sites of PF–Purkinje cell synapses. Finally, a subcellular fractionation analysis revealed that recombinant Cbln1 bound to the synaptosomal and postsynaptic density fractions. These results indicate that Cbln1, released from granule cells as hexamers, specifically binds to a putative receptor located at the postsynaptic sites of PF–Purkinje cell synapses, where it induces synaptogenesis.