Neuroligin-4 Regulates Excitatory Synaptic Transmission in Human Neurons

The autism-associated synaptic-adhesion gene Neuroligin-4 (NLGN4) is poorly conserved evolutionarily, limiting conclusions from Nlgn4 mouse models for human cells. Here, we show that the cellular and subcellular expression of human and murine Neuroligin-4 differ, with human Neuroligin-4 primarily expressed in cerebral cortex and localized to excitatory synapses. Overexpression of NLGN4 in human neurons resulted in an increase in excitatory synapse numbers but a remarkable decrease in synaptic strength. Human neurons carrying the syndromic autism mutation NLGN4-R704C also formed more excitatory synapses but with increased functional synaptic transmission due to a postsynaptic mechanism, while genetic loss of NLGN4 did not significantly affect synapses in the human neurons analyzed. Thus, the NLGN4-R704C mutation represents a change of function mutation. Our work reveals contrasting roles of NLGN4 in human and mouse neurons, suggesting human evolution has impacted even fundamental cell biological processes generally assumed to be highly conserved. We compared the global gene expression profiles of induced neuronal (iN) cells. iN cells were derived from human embryonic stem cells (H1) by expression of transcription factors. Ngn2 was used to generate Ngn2-iN, Alsc1 and Dlx2 to generate AD-iN cells and Alsc1 and Myt1l to generate AM iN cells. AM and Ngn2 iN cells are glutamatergic neurons whereas AD iN cells are GABAergic. Mature iN cells cultured with mouse glia (6 weeks after transgene induction) were dissociated with Trypsin and FACS-sorted in Trizol LS. To distinguish from glial cells, iN cells were transduced with a lentiviral vector expressing EGFP under the control of CAG promoter together with the reprogramming transcription factors.

与自闭症相关的突触黏附基因神经连接蛋白-4（Neuroligin-4, NLGN4）在进化上的保守性较差，这限制了依托Nlgn4小鼠模型所获得的人类细胞相关研究结论。本研究证实，人类与小鼠神经连接蛋白-4的细胞及亚细胞表达模式存在显著差异：人类神经连接蛋白-4主要在大脑皮层中表达，且定位于兴奋性突触。在人类神经元中过表达NLGN4会导致兴奋性突触数量增加，但突触强度却出现显著降低。携带综合征性自闭症突变NLGN4-R704C的人类神经元同样形成了更多的兴奋性突触，但由于突触后机制，其功能性突触传递反而增强；而在本研究分析的人类神经元中，NLGN4的基因缺失并未对突触产生显著影响。由此可见，NLGN4-R704C突变属于功能改变型突变。本研究揭示了NLGN4在人类与小鼠神经元中的作用存在显著差异，这表明人类进化甚至影响了此前普遍认为高度保守的基础细胞生物学过程。我们对诱导神经元（induced neuronal, iN）细胞的全局基因表达谱进行了对比分析。iN细胞通过转录因子介导的表达体系由人类胚胎干细胞（H1）诱导获得：其中，利用Ngn2诱导生成Ngn2-iN细胞，利用Alsc1与Dlx2诱导生成AD-iN细胞，利用Alsc1与Myt1l诱导生成AM iN细胞。AM与Ngn2 iN细胞属于谷氨酸能神经元，而AD iN细胞为γ-氨基丁酸能神经元。将与小鼠胶质细胞共培养的成熟iN细胞（转基因诱导后6周）用胰蛋白酶（Trypsin）消化后，在Trizol LS试剂中进行荧光激活细胞分选。为了与胶质细胞区分，iN细胞在转导重编程转录因子的同时，还转导了携带以CAG启动子调控增强绿色荧光蛋白（EGFP）表达的慢病毒载体。