CRISPR/Cas9 facilitates investigation of neural circuit disease using human iPSCs: mechanism of epilepsy caused by an SCN1A loss-of-function mutation.
CRISPR/Cas9 facilitates investigation of neural circuit disease using human iPSCs: mechanism of epilepsy caused by an SCN1A loss-of-function mutation.
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CRISPR/Cas9 促进使用人类 iPSC 对神经回路疾病的研究：SCN1A 功能丧失突变引起的癫痫机制。
DOI：
10.1038/tp.2015.203
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发表时间：
2016-01-05
影响因子：
6.8
通讯作者：
Li Z
中科院分区：
文献类型：
--
作者：
Liu J;Gao C;Chen W;Ma W;Li X;Shi Y;Zhang H;Zhang L;Long Y;Xu H;Guo X;Deng S;Yan X;Yu D;Pan G;Chen Y;Lai L;Liao W;Li Z
编码Nav1.1通道α亚基的基因SCN 1A的突变可导致具有广泛临床表型的癫痫，其与通道功能丧失或功能获得的对比效应相关。本项目将CRISPR/Cas9和TALEN介导的基因组编辑技术应用于基于诱导多能干细胞（iPSC）的疾病模型，以探讨SCN 1A功能缺失突变导致癫痫的机制。通过使用CRISPR/Cas9荧光标记iPSC衍生的神经元中的GABA能亚型，我们首次对表达SCN 1A的神经亚型进行了电生理学研究，并监测了抑制型和兴奋型的突触后活动。我们发现，突变c.A5768G，导致在外源转染系统中没有Nav1.1的电流，不仅影响Nav1.1表达的GABA能神经元中Nav1.1电流量的性质，而且影响Nav1.1激活的性质。Nav的两种改变进一步降低了患者源性GABA能神经元中动作电位的幅度并提高了阈值，并导致患者源性神经元网络中自发抑制性突触后电流（sIPSC）减弱。虽然自发兴奋性突触后电流（sEPSC）没有显著变化，但当进一步分析sIPSC和sEPSC的频率时，我们发现在患者源性神经元网络中，整个突触后活动从抑制主导状态转移到兴奋状态，这表明仅sIPSC的变化就足以显著逆转自发突触后活动的兴奋水平。总之，我们的发现填补了我们关于SCN 1A突变效应在外源转染细胞和表达Nav1.1的神经元上记录的关系的知识的差距，并揭示了SCN 1A功能丧失突变引起的癫痫发生的生理基础。
Mutations in SCN1A, the gene encoding the α subunit of Nav1.1 channel, can cause epilepsies with wide ranges of clinical phenotypes, which are associated with the contrasting effects of channel loss-of-function or gain-of-function. In this project, CRISPR/Cas9- and TALEN-mediated genome-editing techniques were applied to induced pluripotent stem cell (iPSC)-based-disease model to explore the mechanism of epilepsy caused by SCN1A loss-of-function mutation. By fluorescently labeling GABAergic subtype in iPSC-derived neurons using CRISPR/Cas9, we for the first time performed electrophysiological studies on SCN1A-expressing neural subtype and monitored the postsynaptic activity of both inhibitory and excitatory types. We found that the mutation c.A5768G, which led to no current of Nav1.1 in exogenously transfected system, influenced the properties of not only Nav current amount, but also Nav activation in Nav1.1-expressing GABAergic neurons. The two alterations in Nav further reduced the amplitudes and enhanced the thresholds of action potential in patient-derived GABAergic neurons, and led to weakened spontaneous inhibitory postsynaptic currents (sIPSCs) in the patient-derived neuronal network. Although the spontaneous excitatory postsynaptic currents (sEPSCs) did not change significantly, when the frequencies of both sIPSCs and sEPSCs were further analyzed, we found the whole postsynaptic activity transferred from the inhibition-dominated state to excitation in patient-derived neuronal networks, suggesting that changes in sIPSCs alone were sufficient to significantly reverse the excitatory level of spontaneous postsynaptic activity. In summary, our findings fill the gap of our knowledge regarding the relationship between SCN1A mutation effect recorded on exogenously transfected cells and on Nav1.1-expressing neurons, and reveal the physiological basis underlying epileptogenesis caused by SCN1A loss-of-function mutation.
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影响因子：
3.5
作者：
Ogiwara I;Iwasato T;Miyamoto H;Iwata R;Yamagata T;Mazaki E;Yanagawa Y;Tamamaki N;Hensch TK;Itohara S;Yamakawa K
通讯作者：
Yamakawa K
影响因子：
17.3
作者：
Gaj, Thomas;Gersbach, Charles A.;Barbas, Carlos F., III
通讯作者：
Barbas, Carlos F., III
在白蛋白中间神经元中SCN1a的优先失活会增加癫痫发作的敏感性。
影响因子：
6.1
作者：
Dutton SB;Makinson CD;Papale LA;Shankar A;Balakrishnan B;Nakazawa K;Escayg A
通讯作者：
Escayg A
DOI：
10.1126/science.1232033
发表时间：
2013-02-15
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Mali P;Yang L;Esvelt KM;Aach J;Guell M;DiCarlo JE;Norville JE;Church GM
通讯作者：
Church GM
影响因子：
5.2
作者：
Pankratz, Matthew T.;Li, Xue-Jun;Zhang, Su-Chun
通讯作者：
Zhang, Su-Chun