Evaluation of Scn8a as a target for the treatment of refractory epilepsy

描述（由申请人提供）：癫痫是一种中枢神经系统过度兴奋和同步性疾病，影响全球约5000万人。大约30%的癫痫患者在目前可用的治疗方法下无法充分控制癫痫发作。此外，在过去的40年中，具有治疗抵抗性癫痫发作的患者的百分比没有显著变化，这表明持续需要为难治性癫痫患者确定新的治疗选择。电压门控钠通道（VGSC）SCN1A、SCN2A和SCN3A的突变与几种癫痫亚型相关，包括遗传性（全身性）癫痫伴热性惊厥+（GEFS+）和Dravet综合征（DS）。与此形成鲜明对比的是，我们观察到VGSC Scn8a突变小鼠的癫痫发作抗性增加。此外，Scn8a突变的共分离足以恢复作为DS模型的Scn1a敲除小鼠的正常癫痫发作阈值和寿命，DS是一种灾难性的早期难治性癫痫综合征。这些发现提高了选择性靶向SCN8A治疗难治性癫痫患者的可能性。然而，我们以前的实验的一个局限性是，小鼠从出生就表达了Scn1a和Scn8a突变，因此保护作用可能部分是由于发育过程中的代偿性变化。由于癫痫患者仅在癫痫发作后寻求治疗，因此重要的是确定癫痫发作后降低Scn8a表达水平是否也会改善癫痫发作的严重程度。因此，该提议将研究在癫痫发作后和癫痫发生期间减少Scn8a表达的效果。将具有floxed Scn8a等位基因的小鼠与他莫昔芬诱导型Cre系杂交，以便在成年小鼠中给予他莫昔芬后不可逆地缺失Scn8a。目的1将使用免疫细胞化学来评估在小鼠前脑中表达Scn8a和Scn1a的细胞类型，并将检查在成年、癫痫、Scn1a敲除小鼠中降低Scn8a表达的效果。目的2将确定减少Scn8a表达在颞叶癫痫（TLE）（难治性癫痫的最常见形式）小鼠模型中的作用。海马内注射红藻氨酸可诱发颞叶癫痫发作。目的2将提供有关Scn8a在预防癫痫发生（自发性癫痫发作的过程）中的作用以及在癫痫发作后减少Scn8a的作用的重要信息。将通过脑电图分析测量癫痫发作活动，并进行组织学检查以评估海马形态学变化。这项研究将促进我们对VGSC在癫痫中的作用的理解，并进一步研究靶向Scn8a作为难治性癫痫治疗的可行性。这些结果将具有重要的临床意义，并将促进更多的翻译研究。 公共卫生相关性：癫痫影响全球约5000万人，约30%的癫痫患者对目前可用的治疗没有反应，这表明持续需要开发新的治疗方法。该提案将研究靶向电压门控钠通道基因Scn8a在难治性癫痫中的治疗潜力。)

DESCRIPTION (provided by applicant): Epilepsy is a disorder of central nervous system hyperexcitability and synchrony that affects an estimated 50 million people worldwide. Approximately 30% of epileptic patients do not achieve sufficient control of their seizures with currently available treatments. Moreover, the percentage of patients that have treatment-resistant seizures has not significantly changed in the last 40 years indicating a continued need to identify novel treatment options for patients with refractory epilepsy. Mutations in the voltage gated sodium channels (VGSCs) SCN1A, SCN2A, and SCN3A are associated with several epilepsy subtypes, including genetic (generalized) epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome (DS). In striking contrast, we have observed increased seizure resistance in mice with mutations in the VGSC Scn8a. Furthermore, the co- segregation of an Scn8a mutation was sufficient to restore normal seizure thresholds and lifespans to Scn1a knockout mice that serve as a model of DS, which is a catastrophic, early-life refractory epilepsy syndrome. These findings raise the possibility that selective targeting of SCN8A might be therapeutic in patients with refractory epilepsy. However, one limitation with our previous experiments was that mice expressed the Scn1a and Scn8a mutations from birth, and consequently the protective effect may, in part, have been due to compensatory changes during development. Since patients with epilepsy only seek treatment after seizure onset, it is important to establish whether reducing Scn8a expression levels after seizure onset will also ameliorate seizure severity. As a result, this proposal will investigate the effect of reducing Scn8a expression after seizure onset and during epileptogenesis. Mice with a floxed Scn8a allele will be crossed to a tamoxifen-inducible Cre line in order to irreversibly delete Scn8a following tamoxifen administration in adult mice. Aim 1 will use immunocytochemistry to evaluate the cell types that express Scn8a and Scn1a in the mouse forebrain and will examine the effect of reducing Scn8a expression in adult, epileptic, Scn1a knockout mice. Aim 2 will determine the effect of reducing Scn8a expression in a mouse model of temporal lobe epilepsy (TLE), the most common form of refractory epilepsy. Seizures that originate in the temporal lobe will be induced by intra-hippocampal injection of kainic acid. Aim 2 will provide important information about the role of Scn8a in preventing epileptogenesis (the process by which spontaneous seizures arise) and the effect of reducing Scn8a after seizure onset. Seizure activity will be measured by electroencephalographic analysis and histology will be performed to assess changes in hippocampal morphology. This study will advance our understanding of the role of VGSCs in epilepsy and further examine the feasibility of targeting Scn8a as a treatment for refractory epilepsy. These results will have important clinical implications and will facilitate additional translational studies. PUBLIC HEALTH RELEVANCE: Epilepsy affects an estimated 50 million people worldwide and approximately 30% of epileptic patients do not respond to currently available treatments, indicating a continued need for the development of new therapeutics. This proposal will investigate the therapeutic potential of targeting the voltage-gated sodium channel gene Scn8a in treatment-resistant forms of epilepsy. )