Cbln1 binds to specific postsynaptic sites at parallel fiber–Purkinje cell synapses in the cerebellum
Cbln1 binds to specific postsynaptic sites at parallel fiber–Purkinje cell synapses in the cerebellum
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DOI：
10.1111/j.1460-9568.2009.06639.x
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发表时间：
2009-02
影响因子：
3.4
通讯作者：
K. Matsuda;T. Kondo;Takatoshi Iijima;S. Matsuda;Masahiko Watanabe;M. Yuzaki
中科院分区：
文献类型：
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作者：
K. Matsuda;T. Kondo;Takatoshi Iijima;S. Matsuda;Masahiko Watanabe;M. Yuzaki
Cbln 1属于C1 q/肿瘤坏死因子超家族，是一种独特的分子，它不仅是维持正常的平行纤维（PF）-浦肯野细胞突触所必需的，而且还能够在成人小脑中诱导新的PF突触。虽然据报道Cbln 1是从颗粒细胞中释放出来的，但Cbln 1在小脑中的结合位置和方式在很大程度上仍然不清楚，部分原因是Cbln 1经过蛋白水解产生各种片段，这些片段可以被不同的抗体区别地检测到。为了解决这个问题，我们使用重组Cbln 1对Cbln 1结合位点进行了表征。免疫组化分析显示，重组Cbln 1优先结合PF浦肯野细胞突触在原代培养和急性切片制备中的饱和和可替换的方式。观察到形成六聚体的完整Cbln 1的特异性结合，但未观察到与其他蛋白融合的Cbln 1的N末端或C末端片段的特异性结合。类似地，已经形成三聚体的突变体Cbln 1不与浦肯野细胞结合。免疫反应性的重组Cbln 1中观察到织小脑（缺乏颗粒细胞），但在PCD小脑（缺乏浦肯野细胞），这表明结合位点位于突触后的PF-浦肯野细胞突触的网站上是不存在的。最后，亚细胞分级分析显示，重组Cbln 1绑定到突触体和突触后密度部分。这些结果表明，Cbln 1，从颗粒细胞释放的六聚体，特异性结合到一个假定的受体位于突触后的PF浦肯野细胞突触，在那里它诱导突触发生的网站。
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.