α-neurexins are essential and highly expressed presynaptic cell-adhesion molecules that are frequently linked to neuropsychiatric and neurodevelopmental disorders. Despite their importance, how the elaborate extracellular sequences of α-neurexins contribute to synapse function is poorly understood. We recently characterized the presynaptic gain-of-function phenotype caused by a missense mutation in an evolutionarily conserved extracellular sequence of neurexin-3α (A687T) that we identified in a patient diagnosed with profound intellectual disability and epilepsy. The striking A687T gain-of-function mutation on neurexin-3α prompted us to systematically test using mutants whether the presynaptic gain-of-function phenotype is a consequence of the addition of side-chain bulk (i.e., A687V) or polar/hydrophilic properties (i.e., A687S). We used multidisciplinary approaches in mixed-sex primary hippocampal cultures to assess the impact of the neurexin-3αA687 residue on synapse morphology, function and ligand binding. Unexpectedly, neither A687V nor A687S recapitulated the neurexin-3α A687T phenotype. Instead, distinct from A687T, molecular replacement with A687S significantly enhanced postsynaptic properties exclusively at excitatory synapses and selectively increased binding to neuroligin-1 and neuroligin-3 without changing binding to neuroligin-2 or LRRTM2. Importantly, we provide the first experimental evidence supporting the notion that the position A687 of neurexin-3α and the N-terminal sequences of neuroligins may contribute to the stability of α-neurexin-neuroligin-1 trans-synaptic interactions and that these interactions may specifically regulate the postsynaptic strength of excitatory synapses.Significance Statement Although neurexins were discovered over 30 years ago, our understanding of how the complex extracellular sequences unique to α-neurexins participate in synapse function remains incomplete. We leveraged a previously studied human missense mutation, located in a conserved extracellular region of neurexin-3α and linked to profound intellectual disability and epilepsy, to systematically assess the tolerance of neurexin-3α function to mutations within this region. Using molecular replacement, we assessed how single amino acid substitutions in this extracellular region alters synapse morphology, presynaptic calcium dynamics, and synaptic transmission. We reveal that multiple neurexin ligands unexpectedly use this region to modulate trans-synaptic binding and that different amino acid substitutions in place of the disease mutation result in dramatically different changes to synaptic transmission.

中文翻译：

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对人类 Neurexin-3α 突变的系统结构功能表征揭示了一种细胞外调节序列，该序列可稳定 Neuroligin-1 结合，从而增强兴奋性突触的突触后特性。


α-神经毒素是重要且高度表达的突触前细胞粘附分子，通常与神经精神和神经发育障碍有关。尽管α-神经毒素的复杂细胞外序列很重要，但人们对突触功能的贡献却知之甚少。我们最近描述了由进化上保守的神经毒素-3α (A687T) 细胞外序列中的错义突变引起的突触前功能获得表型，我们在一名被诊断患有严重智力障碍和癫痫的患者中发现了这种错义突变。 neurexin-3α 上引人注目的 A687T 功能获得性突变促使我们使用突变体系统地测试突触前功能获得性表型是否是添加侧链块（即 A687V）或极性/亲水特性的结果（即A687S）。我们在混合性别原代海马培养物中使用多学科方法来评估 neurexin-3αA687 残基对突触形态、功能和配体结合的影响。出乎意料的是，A687V 和 A687S 都没有重现 neurexin-3α A687T 表型。相反，与 A687T 不同，A687S 的分子替代显着增强了兴奋性突触的突触后特性，并选择性增加了与 Neuroligin-1 和 Neuroligin-3 的结合，而不改变与 Neuroligin-2 或 LRRTM2 的结合。重要的是，我们提供了第一个实验证据，支持这样的观点，即 neurexin-3α 的位置 A687 和 Neuroligin 的 N 端序列可能有助于 α-neurexin-neuroligin-1 跨突触相互作用的稳定性，并且这些相互作用可能具体调节兴奋性突触的突触后强度。意义陈述 尽管神经毒素在 30 多年前就被发现了，但我们对 α-神经毒素独特的复杂细胞外序列如何参与突触功能的理解仍然不完整。我们利用先前研究的人类错义突变（位于 neurexin-3α 的保守细胞外区域，与严重的智力障碍和癫痫相关）来系统地评估 neurexin-3α 功能对该区域内突变的耐受性。使用分子替换，我们评估了该细胞外区域中的单个氨基酸替换如何改变突触形态、突触前钙动力学和突触传递。我们揭示了多个神经毒素配体出乎意料地使用该区域来调节跨突触结合，并且代替疾病突变的不同氨基酸取代导致突触传递发生显着不同的变化。