神経ネットワーク形成における微小管結合タンパク質タウのアイソフォーム特異的な役割

微管相关蛋白 tau 在神经网络形成中的异构体特异性作用

基本信息

批准号：
19J13396
负责人：
DILINA Tuerde
金额：
$ 1.34万
依托单位：
Keio University (2020)Tokyo Metropolitan University (2019)
依托单位国家：
日本
项目类别：
Grant-in-Aid for JSPS Fellows
财政年份：
2019
资助国家：
日本
起止时间：
2019-04-25 至 2021-03-31
项目状态：
已结题

项目摘要

To clarify the tau isoform specific function in neuronal connection in vivo, I tried to apply the GRAPHIC system to label all neuron to neuron connections which is new technique based on biomolecular fluorescence complementation (BiFC) and has been developed to label all neurons connections.Firstly, to determine whether the GRAPHIC system could able to detect all neuronal connections that included both synaptic and non-synaptic, I applied the GRAPHIC system to the dopamine mesocorticolimbic pathway, the input from the ventral tegmental area (VTA) into nucleus accumbens (NAc), because of previous studies assumed that dopamine neurons could be formed both synaptic and non-synaptic communication with other neurons. Dopamine axons of VTA labeled by expressing C- GRAPHIC binds with AAV- EF1a-DIO-mCherry and co-expressing with AAV-TH-Cre, and neurons in NAc labeled by expressing N- GRAPHIC-mTagBFP2 with hSynapsinI promoter. After one month virus post-infection, I confirmed that the GRAPHIC signal expressed under mCherry (the source of dopamine neuron) and blue mTagBFP2 (the target neuron) colors as well as expressed with or without postsynaptic density, which indicated the GRAPHIC system could successfully label synaptic and non-synaptic connections sites.During this year, I tried to re-design the GRAPHIC system for my experimental propose and tested its efficiency in vivo. By establishing the GRAPHIC system in the dopaminergic pathway, I showed the evidence for the first time the dopamine neurons make both synaptic and non-synaptic interactions with other neurons.

为了阐明体内神经元连接中 tau 亚型的特定功能，我尝试应用 GRAPHIC 系统来标记所有神经元与神经元的连接，这是基于生物分子荧光互补 (BiFC) 的新技术，已开发用于标记所有神经元连接。，为了确定 GRAPHIC 系统是否能够检测包括突触和非突触的所有神经元连接，我将 GRAPHIC 系统应用于多巴胺中皮质边缘通路，从腹侧被盖区（VTA）输入到伏隔核（NAc），因为之前的研究假设多巴胺神经元可以与其他神经元形成突触和非突触通讯。通过表达 C-GRAPHIC 标记的 VTA 多巴胺轴突与 AAV-EF1a-DIO-mCherry 结合并与 AAV-TH-Cre 共表达，以及通过用 hSynapsinI 启动子表达 N-GRAPHIC-mTagBFP2 标记的 NAc 中的神经元。病毒感染一个月后，我确认GRAPHIC信号在mCherry（多巴胺神经元来源）和蓝色mTagBFP2（目标神经元）颜色下表达，以及有或没有突触后密度表达，这表明GRAPHIC系统可以成功标记突触和非突触连接位点。今年，我尝试为我的实验方案重新设计图形系统，并在体内测试其效率。通过在多巴胺能通路中建立图形系统，我首次展示了多巴胺神经元与其他神经元进行突触和非突触相互作用的证据。