Department of Brain and Cognitive Sciences, Daegu Gyeongbuk Institute of Science and Technology, Daegu 42988, Korea.
Department of Molecular and Cellular Physiology, Shinshu University School of Medicine, Matsumoto 390-8621, Japan.
J Neurosci. 2018 Jul 25;38(30):6700-6721. doi: 10.1523/JNEUROSCI.0672-18.2018. Epub 2018 Jun 22.
Leukocyte common antigen-receptor protein tyrosine phosphatases (LAR-RPTPs) are hub proteins that organize excitatory and inhibitory synapse development through binding to various extracellular ligands. Here, we report that knockdown (KD) of the LAR-RPTP family member PTPσ reduced excitatory synapse number and transmission in cultured rat hippocampal neurons, whereas KD of PTPδ produced comparable decreases at inhibitory synapses, in both cases without altering expression levels of interacting proteins. An extensive series of rescue experiments revealed that extracellular interactions of PTPσ with Slitrks are important for excitatory synapse development. These experiments further showed that the intracellular D2 domain of PTPσ is required for induction of heterologous synapse formation by Slitrk1 or TrkC, suggesting that interaction of LAR-RPTPs with distinct intracellular presynaptic proteins, drives presynaptic machinery assembly. Consistent with this, double-KD of liprin-α2 and -α3 or KD of PTPσ substrates (N-cadherin and p250RhoGAP) in neurons inhibited Slitrk6-induced, PTPσ-mediated heterologous synapse formation activity. We propose a synaptogenesis model in presynaptic neurons involving LAR-RPTP-organized retrograde signaling cascades, in which both extracellular and intracellular mechanisms are critical in orchestrating distinct synapse types. In this study, we sought to test the unproven hypothesis that PTPσ and PTPδ are required for excitatory and inhibitory synapse formation/transmission, respectively, in cultured hippocampal neurons, using knockdown-based loss-of-function analyses. We further performed extensive structure-function analyses, focusing on PTPσ-mediated actions, to address the mechanisms of presynaptic assembly at excitatory synaptic sites. Using interdisciplinary approaches, we systematically applied a varied set of PTPσ deletion variants, point mutants, and splice variants to demonstrate that both extracellular and intracellular mechanisms are involved in organizing presynaptic assembly. Strikingly, extracellular interactions of PTPσ with heparan sulfates and Slitrks, intracellular interactions of PTPσ with liprin-α and its associated proteins through the D2 domain, as well as distinct substrates are all critical.
白细胞共同抗原受体蛋白酪氨酸磷酸酶（LAR-RPTPs）是枢纽蛋白，通过与各种细胞外配体结合，组织兴奋性和抑制性突触的发育。在这里，我们报告说 LAR-RPTP 家族成员 PTPσ 的敲低（KD）减少了培养的大鼠海马神经元中的兴奋性突触数量和传递，而 PTPδ 的 KD 在抑制性突触中产生了类似的减少，在这两种情况下都没有改变相互作用蛋白的表达水平。一系列广泛的挽救实验表明，PTPσ 与 Slitrks 的细胞外相互作用对于兴奋性突触发育很重要。这些实验进一步表明，PTPσ 的细胞内 D2 结构域对于 Slitrk1 或 TrkC 诱导的异源突触形成是必需的，这表明 LAR-RPTPs 与不同的细胞内突触前蛋白的相互作用，驱动突触前机制的组装。与此一致的是，神经元中 liprin-α2 和 -α3 的双重 KD 或 PTPσ 底物（N-钙粘蛋白和 p250RhoGAP）的 KD 抑制了 Slitrk6 诱导的、由 PTPσ 介导的异源突触形成活性。我们提出了一个在突触前神经元中的突触发生模型，涉及 LAR-RPTP 组织的逆行信号级联，其中细胞外和细胞内机制在协调不同的突触类型方面都是至关重要的。在这项研究中，我们试图通过基于敲低的功能丧失分析来测试未经证实的假设，即 PTPσ 和 PTPδ 分别是培养的海马神经元中兴奋性和抑制性突触形成/传递所必需的。我们进一步进行了广泛的结构-功能分析，重点是 PTPσ 介导的作用，以解决兴奋性突触位点的突触前组装机制。我们系统地应用了一系列不同的 PTPσ 缺失变体、点突变体和剪接变体，通过跨学科方法，利用各种 PTPσ 缺失变体、点突变体和剪接变体，证明了细胞外和细胞内机制都参与了突触前组装的组织。引人注目的是，PTPσ 与肝素硫酸盐和 Slitrks 的细胞外相互作用、PTPσ 与 liprin-α 及其相关蛋白通过 D2 结构域的细胞内相互作用，以及不同的底物都是至关重要的。
