Department of Physiological Genomics, Biomedical Center, Ludwig-Maximilians-University, Großhaderner Str. 9, D-82152 Planegg-Martinsried, Germany; Institute for Stem Cell Research, German Research Center for Environmental Health, Helmholtz Centre Munich, Ingolstädter Landstraße 1, D-85764 Neuherberg, Germany.
Department of Physiological Genomics, Biomedical Center, Ludwig-Maximilians-University, Großhaderner Str. 9, D-82152 Planegg-Martinsried, Germany.
Neuroscience. 2019 Nov 1;419:60-71. doi: 10.1016/j.neuroscience.2019.09.011. Epub 2019 Oct 28.
Agrin is a multi-domain protein best known for its essential function during formation of the neuromuscular junction. Alternative mRNA splicing at sites named y and z in the C-terminal part of agrin regulates its interaction with a receptor complex consisting of the agrin-binding low-density lipoprotein receptor-related protein 4 (Lrp4) and the muscle-specific kinase (MuSK). Isoforms with inserts at both splice sites bind to Lrp4, activate MuSK and are synaptogenic at the neuromuscular junction. Agrin is also expressed as a transmembrane protein in the central nervous system (CNS) but its function during interneuronal synapse formation is unclear. Recently we demonstrated that transfection of a full-length cDNA coding for transmembrane agrin (TM-agrin) in cultured embryonic cortical neurons induced an Lrp4-dependent but MuSK-independent increase in dendritic glutamatergic synapses and an Lrp4- and MuSK-independent reduction of inhibitory synapses. Here we show that presynaptic specializations were similarly affected by TM-agrin overexpression. In addition, we mapped the regions within TM-agrin responsible for TM-agrin's effects on dendritic aggregates of synapse-associated proteins. We show that the presence of a four amino acid insert at splice site y is essential for the increase in the density of puncta containing the postsynaptic density protein 95 kDa. This effect was independent of splice site z. The reduction of the gephyrin puncta density was independent of the entire extracellular part of TM-agrin but required a highly conserved serine residue in the intracellular domain of TM-agrin. These results provide further evidence for a function of TM-agrin during CNS synaptogenesis and demonstrate that different domains and alternative splicing of TM-agrin differentially affect excitatory and inhibitory synapse formation in cultured embryonic CNS neurons.
聚集蛋白是一种多功能蛋白，其在形成神经肌肉接头中的作用至关重要。在聚集蛋白 C 端部分的 y 和 z 位点的选择性 mRNA 剪接调节其与包含聚集蛋白结合低密度脂蛋白受体相关蛋白 4（Lrp4）和肌肉特异性激酶（MuSK）的受体复合物的相互作用。在两个剪接位点都插入的同种型与 Lrp4 结合，激活 MuSK，并在神经肌肉接头处具有突触发生作用。聚集蛋白也作为跨膜蛋白在中枢神经系统（CNS）中表达，但在中间神经元突触形成中的功能尚不清楚。最近，我们证明在培养的胚胎皮质神经元中转染编码跨膜聚集蛋白（TM-agrin）的全长 cDNA 可诱导 Lrp4 依赖性但 MuSK 非依赖性的树突状谷氨酸能突触增加和 Lrp4 和 MuSK 非依赖性的抑制性突触减少。在这里，我们表明 TM-agrin 过表达同样影响突触前特化。此外，我们绘制了 TM-agrin 中负责 TM-agrin 对突触相关蛋白树突聚集影响的区域。我们表明，在剪接位点 y 处存在四氨基酸插入对于增加包含突触后密度蛋白 95kDa 的斑点密度是必需的。这种作用与剪接位点 z 无关。Gephyrin 斑点密度的减少与 TM-agrin 的整个细胞外部分无关，但需要 TM-agrin 细胞内结构域中的一个高度保守的丝氨酸残基。这些结果为 TM-agrin 在中枢神经系统突触发生中的功能提供了进一步的证据，并证明 TM-agrin 的不同结构域和选择性剪接不同地影响培养的胚胎中枢神经系统神经元中兴奋性和抑制性突触的形成。
