We recently identified an autism spectrum disorder/intellectual disability (ASD/ID)-related de novo mutation hotspot in the Rac1-activating GEF1 domain of the protein Trio. Trio is a Rho guanine nucleotide exchange factor (RhoGEF) that is essential for glutamatergic synapse function. An ASD/ID-related mutation identified in Trio's GEF1 domain, Trio D1368V, produces a pathologic increase in glutamatergic synaptogenesis, suggesting that Trio is coupled to synaptic regulatory mechanisms that govern glutamatergic synapse formation. However, the molecular mechanisms by which Trio regulates glutamatergic synapses are largely unexplored. Here, using biochemical methods, we identify an interaction between Trio and the synaptogenic protein Neuroligin 1 (NLGN1) in the brain. Molecular biological approaches were then combined with super-resolution dendritic spine imaging and whole-cell voltage-clamp electrophysiology in hippocampal slices from male and female rats to examine the impact ASD/ID-related Trio mutations have on NLGN1-mediated synaptogenesis. We find that an ASD/ID-related mutation in Trio's eighth spectrin repeat region, Trio N1080I, inhibits Trio's interaction with NLGN1 and prevents Trio D1368V-mediated synaptogenesis. Inhibiting Trio's interaction with NLGN1 via Trio N1080I blocked NLGN1-mediated synaptogenesis and increases in synaptic NMDA Receptor function but not NLGN1-mediated increases in synaptic AMPA Receptor function. Finally, we show that the aberrant synaptogenesis produced by Trio D1368V is dependent on NLGN signaling. Our findings demonstrate that ASD/ID-related mutations in Trio are able to pathologically increase as well as decrease NLGN-mediated effects on glutamatergic neurotransmission, and point to an NLGN1-Trio interaction as part of a key pathway involved in ASD/ID etiology.

SIGNIFICANCE STATEMENT A number of genes have been implicated in the development of autism spectrum disorder/intellectual disability (ASD/ID) in humans. It is now important to identify relationships between these genes to uncover specific cellular regulatory pathways that contribute to these disorders. In this study, we discover that two glutamatergic synapse regulatory proteins implicated in ASD/ID, Trio and Neuroligin 1, interact with one another to promote glutamatergic synaptogenesis. We also identify ASD/ID-related mutations in Trio that either inhibit or augment Neuroligin 1-mediated glutamatergic synapse formation. Together, our results identify a synaptic regulatory pathway that, when disrupted, likely contributes to the development of ASD/ID. Going forward, it will be important to determine whether this pathway represents a point of convergence of other proteins implicated in ASD/ID.

我们最近发现了一种与自闭症谱系障碍/智力障碍 (ASD/ID) 相关的从头蛋白 Trio 的 Rac1 激活 GEF1 域中的突变热点。Trio 是一种 Rho 鸟嘌呤核苷酸交换因子 (RhoGEF)，对谷氨酸能突触功能至关重要。在 Trio 的 GEF1 结构域 Trio D1368V 中发现的 ASD/ID 相关突变导致谷氨酸能突触发生的病理性增加，表明 Trio 与控制谷氨酸能突触形成的突触调节机制相结合。然而，Trio 调节谷氨酸能突触的分子机制在很大程度上尚未得到探索。在这里，我们使用生化方法确定了 Trio 和大脑中突触蛋白 Neuroligin 1 (NLGN1) 之间的相互作用。然后将分子生物学方法与来自雄性和雌性大鼠的海马切片中的超分辨率树突棘成像和全细胞电压钳电生理学相结合，以检查 ASD/ID 相关的 Trio 突变对 NLGN1 介导的突触发生的影响。我们发现 Trio 的第八个血影蛋白重复区域 Trio N1080I 中的 ASD/ID 相关突变抑制了 Trio 与 NLGN1 的相互作用并阻止了 Trio D1368V 介导的突触发生。通过 Trio N1080I 抑制 Trio 与 NLGN1 的相互作用会阻止 NLGN1 介导的突触发生并增加突触 NMDA 受体功能，但不会增加 NLGN1 介导的突触 AMPA 受体功能。最后，我们表明 Trio D1368V 产生的异常突触发生依赖于 NLGN 信号。

意义声明许多基因与人类自闭症谱系障碍/智力障碍 (ASD/ID) 的发展有关。现在重要的是确定这些基因之间的关系，以揭示导致这些疾病的特定细胞调节途径。在这项研究中，我们发现与 ASD/ID、Trio 和 Neuroligin 1 相关的两种谷氨酸能突触调节蛋白彼此相互作用以促进谷氨酸能突触发生。我们还确定了 Trio 中与 ASD/ID 相关的突变，这些突变抑制或增强了 Neuroligin 1 介导的谷氨酸能突触形成。总之，我们的结果确定了一种突触调节途径，当该途径被破坏时，可能有助于 ASD/ID 的发展。往前走，