Cbln1 is essential for synaptic integrity and plasticity in the cerebellum Hirokazu Hirai 1,3,4 , Zhen Pang 1,3,4 , Dashi Bao 1 , Taisuke Miyazaki 2 , Leyi Li 1 , Eriko Miura 2 , Jennifer Parris 1 , Yongqi Rong 1 , Masahiko Watanabe 2 , Michisuke Yuzaki 1,3 & James I Morgan 1 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, GluRd2, 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 identification of molecules that influence synapse formation and function has importance for our understanding of the mechanisms contributing to both neurodevelopment and information storage and processing in the nervous system. Moreover, such molecules could potentially contribute to the pathogenesis of neurological and neurop- sychiatric disorders and might represent previously unknown thera- peutic targets or entities. Here we elucidate the properties of Cbln1, a secreted protein that is essential for synapse structure and function in the cerebellum. Cbln1 is the prototype for a family of four brain-specific proteins (Cbln1–Cbln4) of unknown function that was first identified by virtue of its harboring a naturally occurring 16-amino-acid peptide, cere- bellin 1 . Both cbln1 and cbln3 mRNAs are selectively expressed in the cerebellum, and their levels increase in parallel with synaptogenesis 2 . As the cerebellin peptide is enriched in synaptosomes 3 and has been reported to undergo release upon depolarization 4 , it was initially suggested to be a neuromodulator. However, Cbln1 lacks the typical structural features of a neuropeptide precursor; rather, it belongs to the C1q and tumor necrosis factor (TNF) family 4 . The members of this family are frequently secreted as trimers that serve diverse roles in intercellular communication. Indeed, TNFa has been implicated in neuronal plasticity 5 . We show here that Cbln1 is secreted from cerebellar granule cells as a glycoprotein that controls synaptic plasticity and synapse integrity of Purkinje cells. RESULTS Although cbln1 mRNA is enriched in adult cerebellum 1 , the source of Cbln1 is unknown. In situ hybridization showed that, like cbln3 (ref. 2), cbln1 mRNA was localized to the internal granule cell layer (Fig. 1a,b). Owing to the limited resolution of in situ hybridization, we could not conclusively exclude some expression of cbln1 mRNA within Purkinje cells, which is notable for three reasons: the cerebellin peptide is en- riched in synaptosomes 3 , anti-cerebellin antibodies react with Purkinje cell dendritic spines 6 and the deficits described below in cbln1-null mice involve Purkinje cells. Therefore, we generated transgenic mice in which b-galactosidase was driven from the mouse cbln1 promoter. Analysis of cbln1-lacZ mice demonstrated expression of the reporter in granule cells but not Purkinje cells (Fig. 1c,d). Using in situ hybridization and cbln1-lacZ mice, we did not observe cbln1 expression in the inferior olive, which sends climbing fiber (CF) inputs to Purkinje cells (Supplementary Fig. 1). Thus, Cbln1 may be secreted from granule cells and interact with postsynaptic structures of Purkinje cells. To establish whether Cbln1 was secreted, we performed immuno- blotting. Immunoblotting of culture medium of cerebellar granule cells identified a protein of approximately 35 kDa, larger than the predicted molecular weight for Cbln1 (B20 kDa; Fig. 1e). An immunoreactive protein of equivalent mass was also detected in medium from HEK 293 cells expressing recombinant Cbln1 (Fig. 1f ) but not empty vector (data not shown). Moreover, an immunoreactive band with the same pattern of migration as secreted Cbln1 was detected in cerebellar extracts (Fig. 1g) indicating that it also exists in vivo. Treatment of secreted recombinant Cbln1 with glycosidases con- verted the 35 kDa band into two products of lesser mass (Fig. 1f ), suggesting glycosylation. Furthermore, treatment of cerebellar lysates with glycosidases eliminated the 35 kDa band and generated bands Received 13 July 2005; accepted 21 September 2050; published online 23 October 2005; doi:10.1038/nn1576 1 Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, 332 North Lauderdale Street, MS 323, Memphis, Tennessee 38105-2794, USA. 2 Department of Anatomy, Hokkaido University School of Medicine, Sapporo 060-8638, Japan. 3 Current addresses: Advanced Science Research Center, Kanazawa University, PRESTO, Japan Science and Technology Agency, Kanazawa 920-8640, Japan (H.H.); Roche Palo Alto, 3431 Hillview Ave., R2-101 Palo Alto, California 94304- 1320 USA (Z.P.) and Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan (M.Y.) 4 These authors contributed equally to this work. Correspondence should be addressed to M.Y. (myuzaki@sc.itc.keio.ac.jp) or J.M. (jim.morgan@stjude.org). 1534 VOLUME 8 [ NUMBER 11 [ NOVEMBER 2005 NATURE NEUROSCIENCE ARTICLES © 2005 Nature Publishing Group http://www.nature.com/natureneuroscience