SUDEP Research Alliance: iPSC and Mouse Neurocardiac Models, Application 6 of 7

 描述（由申请人提供）：本SUDEP研究联盟无墙中心（CWOW）资助提案的申请6，“iPSC和小鼠神经心脏模型”，探讨了心律失常和自主神经功能障碍作为SUDEP的潜在原因。尽管SUDEP是癫痫最具破坏性的后果，也是癫痫死亡的主要原因，但人们对其原因了解甚少，也没有生物标志物来识别有风险的癫痫患者。为了促进我们对这些关键问题的理解，我们将重点关注Dravet综合征（DS），这是一种与高SUDEP发病率相关的严重儿童癫痫性脑病。DS最常由编码NaV1.1的电压门控Na+通道（VGSC）基因SCN 1A突变引起。由于NaV1.1在脑、心脏和外周神经中表达，一个令人信服的想法是，除了中枢神经元外，DS心肌细胞（CM）或自主神经元中的Na+电流（INa）改变会导致心律失常和SUDEP。我们使用诱导多能干细胞（iPSC）方法从DS受试者的成纤维细胞中获得中枢和外周神经元和CM。DS患者CM的初步数据表明，DS受试者的一个子集显示CM INa和兴奋性异常。在DS人突变SCN 1A基因敲入小鼠模型的研究中，我们观察到自发性癫痫发作和SUDEP、心室CM INa密度增加和室性心律失常。同样，我们在Scn 1b缺失DS小鼠模型中发现心室CM INa密度增加、自发性癫痫发作和SUDEP。我们的工作，Scn 1a杂合子无效DS小鼠的研究，以及DS的临床ECG研究也显示心脏自主神经功能改变。因此，我们假设DS中的SUDEP是由VGSC突变引起的，除了脑功能障碍外，VGSC突变还产生心脏电和/或自主神经功能障碍。此外，结合DS患者来源的细胞、小鼠模型和患者发作期ECG数据的研究，将产生DS中SUDEP风险的生物标志物。 四个具体目标将测试这些假设：1）使用DS患者iPSC衍生的CM和DS小鼠来理解DS连锁的SCN 1A突变对心脏兴奋性的影响; 2）使用DS患者iPSC衍生的自主神经元和DS小鼠来确定DS连锁的SCN 1A突变如何影响自主神经元的兴奋性、心脏自主神经支配和心脏功能的自主控制; 3）研究DS的第二小鼠模型、Scn 1b缺失小鼠和SCN 1B-DS患者iPSC CM和神经元中自主兴奋性的变化;和4）确定DS患者在基线或发作期的心脏电和/或自主功能是否改变。我们的工作将与整个CWOW提案协同作用，不仅揭示DS中的SUDEP机制，而且还提供了解SUDEP原因和生物标志物的进展，这些生物标志物将适用于其他难治性癫痫，因为离子通道病和其他病因。这项工作还将显示使用多个平台（蜂窝）的原理证明 和来自相同患者的临床数据，以及多个小鼠模型），以个体化SUDEP风险并开发患者特异性预防性治疗。

 DESCRIPTION (provided by applicant): Application 6 of this SUDEP Research Alliance Centers Without Walls (CWOW) grant proposal, "iPSC and Mouse Neurocardiac Models," explores cardiac arrhythmia and autonomic dysfunction as potential causes of SUDEP. Although SUDEP is the most devastating consequence of epilepsy and the leading cause of epilepsy mortality, astonishingly little is understood about its causes and no biomarkers exist to identify at risk epilepsy patients. To advance our understanding of these critical issues, we will focus on Dravet Syndrome (DS), a severe childhood epileptic encephalopathy associated with a high SUDEP incidence. DS is most frequently caused by mutations in the voltage-gated Na+ channel (VGSC) gene SCN1A, encoding NaV1.1. As NaV1.1 is expressed in brain, heart, and peripheral nerves, a compelling idea is that altered Na+ currents (INa) in DS cardiac myocytes (CMs) or autonomic neurons, in addition to central neurons, lead to arrhythmias and SUDEP. We used the induced pluripotent stem cell (iPSC) method to derive central and peripheral neurons and CMs from fibroblasts of DS subjects. Preliminary data from DS patient CMs suggest that a subset of DS subjects shows abnormal CM INa and excitability. In studies of a DS human mutant SCN1A knock-in mouse model, we observed spontaneous seizures and SUDEP, increased ventricular CM INa density, and ventricular arrhythmias at the time of SUDEP. Similarly, we found increased ventricular CM INa density, spontaneous seizures and SUDEP in a Scn1b null DS mouse model. Our work, studies of Scn1a heterozygous null DS mice, and clinical ECG studies in DS also show altered cardiac autonomic function. Thus, we hypothesize that SUDEP in DS is caused by VGSC mutations that produce cardiac electrical and/or autonomic dysfunction, in addition to brain dysfunction. Furthermore, that combined insights from studies of DS patient-derived cells, mouse models and patient peri-ictal ECG data will yield biomarkers of SUDEP risk in DS. Four specific aims will test these hypotheses: 1) To understand the effects of DS-linked SCN1A mutations on cardiac excitability using DS patient iPSC-derived CMs and DS mice; 2) To determine how DS-linked SCN1A mutations influence the excitability of autonomic neurons, cardiac autonomic innervation, and autonomic control of cardiac function using DS patient iPSC-derived autonomic neurons and DS mice; 3) To investigate changes in autonomic excitability in a second mouse model of DS, Scn1b null mice, and in SCN1B-DS patient iPSC CMs and neurons; and 4) To determine whether cardiac electrical and/or autonomic function is altered in DS patients at baseline or peri-ictally. Our wor will synergize with the entire CWOW proposal to not only uncover SUDEP mechanisms in DS, but also to provide advances in understanding SUDEP causes and biomarkers that will be applicable to other refractory epilepsies due to ion channelopathies and perhaps other etiologies. This work will also show proof-of-principle for the use of multiple platforms (cellular and clinical data from the same patients, and multiple mouse models) to individualize SUDEP risk and develop patient-specific preventative treatments.