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Abstract：
Synapse is the fundamental structure for neurons to transmit information between cells. The proper synapse formation is crucial for developing neural circuits and cognitive functions of the brain. The aberrant synapse formation has been proved to cause many neurological disorders, including autism spectrum disorders and intellectual disability. Synaptic cell adhesion molecules (CAMs) are thought to play a major role in achieving mechanistic cell-cell recognition and initiating synapse formation via trans-synaptic interactions. Due to the diversity of synapses in different brain areas, circuits and neurons, although many synaptic CAMs, such as Neurexins (NRXNs), Neuroligins (NLGNs), Synaptic cell adhesion molecules (SynCAMs), Leucine-rich-repeat transmembrane neuronal proteins (LRRTMs), and SLIT and NTRK-like protein (SLITRKs) have been identified as synaptogenic molecules, how these molecules determine specific synapse formation and whether other molecules driving synapse formation remain undiscovered are unclear. Here, to provide a tool for synapse labeling and synaptic CAMs screening by artificial synapse formation (ASF) assay, we generated synaptotagmin-1-tdTomato (Syt1-tdTomato) transgenic mice by inserting the tdTomato-fused synaptotagmin-1 coding sequence into the genome of C57BL/6J mice. In the brain of Syt1-tdTomato transgenic mice, the tdTomato-fused synaptotagmin-1 (SYT1-tdTomato) signals were widely observed in different areas and overlapped with synapsin-1, a widely-used synaptic marker. In the olfactory bulb, the SYT1-tdTomato signals are highly enriched in the glomerulus. In the cultured hippocampal neurons, the SYT1-tdTomato signals showed colocalization with several synaptic markers. Compared to the wild-type (WT) mouse neurons, cultured hippocampal neurons from Syt1-tdTomato transgenic mice presented normal synaptic neurotransmission. In ASF assays, neurons from Syt1-tdTomato transgenic mice could form synaptic connections with HEK293T cells expressing NLGN2, LRRTM2, and SLITRK2 without immunostaining. Therefore, our work suggested that the Syt1-tdTomato transgenic mice with the ability to label synapses by tdTomato, and it will be a convenient tool for screening synaptogenic molecules.
摘要：
突触是神经元在细胞间传递信息的根本构造。正确的突触形成对于发展大脑的神经回路和认知功能至关重要。异常的突触形成已被证明会导致许多神经系统疾病，包括自闭症谱系障碍和智力障碍。突触细胞粘附分子（CAM）被认为在实现机械性细胞-细胞识别和通过跨突触相互作用启动突触形成中起主要作用。由于不同大脑区域突触的多样性，电路和神经元，虽然许多突触CAM，例如Neurexins(NRXN)，Neuroligins(NLGNs),突触细胞粘附分子(SynCAM),富含亮氨酸的重复跨膜神经元蛋白（LRRTM）和SLIT和NTRK样蛋白（SLITRK）已被鉴定为突触分子，这些分子如何决定特定的突触形成，以及其他驱动突触形成的分子是否仍未被发现，目前尚不清楚。这里,为通过人工突触形成（ASF）分析进行突触标记和突触CAM筛选提供工具，我们通过将tdTomato融合的synaptotagmin-1编码序列插入C57BL/6J小鼠的基因组中，产生了synaptotagmin-1-tdTomato（Syt1-tdTomato）转基因小鼠。在Syt1-tdTomato转基因小鼠的大脑中，tdTomato融合的突触蛋白-1（SYT1-tdTomato）信号在不同区域广泛观察到，并与广泛使用的突触标记物突触蛋白1重叠。在嗅球中，SYT1-tdTomato信号在肾小球中高度富集。在培养的海马神经元中，SYT1-tdTomato信号显示与几种突触标记的共定位。与野生型（WT）小鼠神经元相比，Syt1-tdTomato转基因小鼠培养的海马神经元表现出正常的突触神经传递。在ASF检测中，Syt1-td番茄转基因小鼠的神经元可以与表达NLGN2，LRRTM2和SLITRK2的HEK293T细胞形成突触连接，无需免疫染色。因此,我们的工作表明，Syt1-tdTomato转基因小鼠具有通过tdTomato标记突触的能力，它将是筛选突触分子的便捷工具。
