Cellular and Subcellular Localization of Endogenous Neuroligin-1 in the Cerebellum.
Cellular and Subcellular Localization of Endogenous Neuroligin-1 in the Cerebellum.
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小脑内源性 Neuroligin-1 的细胞和亚细胞定位。
DOI：
10.1007/s12311-018-0966-x
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发表时间：
2018
期刊：
影响因子：
3.5
通讯作者：
Yuzaki M.
中科院分区：
文献类型：
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作者：
Nozawa K;Hayashi A;Motohashi J;Takeo YH;Matsuda K;Yuzaki M.
突触在整个生命过程中被称为突触组织者的分子精确地建立、维持和修饰，这些分子包括神经毒素和神经配蛋白（Nlgn）。尽管突触组织者在定义神经元回路的功能方面很重要，但由于缺乏用于免疫组织化学研究的特异性抗体，许多突触组织者的细胞和亚细胞定位仍然很难确定。在本研究中，我们没有产生特异性抗体，而是在Nlgn 1基因中插入了血凝素（HA）表位的敲入小鼠。我们通过将CRISPR/Cas9系统递送到受精卵中实现了高通量和精确的基因编辑。使用HA-Nlgn 1小鼠，我们发现HA-Nlgn 1在平行纤维和分子层中间神经元（MLI）与肾小球之间的突触处富集，其中苔藓纤维终末与颗粒细胞树突突触。HA免疫反应共定位与突触后密度95在这些突触，表明内源性Nlgn 1位于兴奋性突触后位点。相反，HA-Nlgn 1信号在浦肯野细胞的树突和胞体中非常弱。有趣的是，HA-免疫反应性也观察到的pinceau，一个专门的结构形成的MLI轴突和星形胶质细胞。HA-免疫反应性的pinceau显着减少敲低Nlgn 1在MLI，表明除了突触后位点，Nlgn 1也定位于MLI轴突。我们的研究结果表明，通过CRISPR/Cas9基于电穿孔的基因编辑进行表位标记是一种可行且强大的方法，用于以亚细胞分辨率绘制内源性突触组织者，而不需要针对每种蛋白质的特异性抗体。
Synapses are precisely established, maintained, and modified throughout life by molecules called synaptic organizers, which include neurexins and neuroligins (Nlgn). Despite the importance of synaptic organizers in defining functions of neuronal circuits, the cellular and subcellular localization of many synaptic organizers has remained largely elusive because of the paucity of specific antibodies for immunohistochemical studies. In the present study, rather than raising specific antibodies, we generated knock-in mice in which a hemagglutinin (HA) epitope was inserted in theNlgn1gene. We have achieved high-throughput and precise gene editing by delivering the CRISPR/Cas9 system into zygotes. UsingHA-Nlgn1mice, we found that HA-Nlgn1 was enriched at synapses between parallel fibers and molecular layer interneurons (MLIs) and the glomeruli, in which mossy fiber terminals synapse onto granule cell dendrites. HA immunoreactivity was colocalized with postsynaptic density 95 at these synapses, indicating that endogenous Nlgn1 is localized at excitatory postsynaptic sites. In contrast, HA-Nlgn1 signals were very weak in dendrites and somata of Purkinje cells. Interestingly, HA-immunoreactivities were also observed in the pinceau, a specialized structure formed by MLI axons and astrocytes. HA-immunoreactivities in the pinceau were significantly reduced by knockdown of Nlgn1 in MLIs, indicating that in addition to postsynaptic sites, Nlgn1 is also localized at MLI axons. Our results indicate that epitope-tagging by electroporation-based gene editing with CRISPR/Cas9 is a viable and powerful method for mapping endogenous synaptic organizers with subcellular resolution, without the need for specific antibodies for each protein.