Elucidating pathogenic mechanisms in STRADA-related brain malformation and epilepsy

项目总结 癫痫是导致发病率、死亡率、残疾和费用的主要原因，影响着#年超过47万儿童。 虽然美国已经开发了许多控制癫痫发作的药物，但大约30%的患者没有 对药物有反应，到目前为止，还没有药物可以防止或阻止疾病的进展 癫痫。最近，许多癫痫的遗传原因已被确定，这为人们提供了对癫痫途径的洞察。 与癫痫的发生有关。导致mTOR通路过度活跃的突变(所谓的mTORpathies) 已成为脑畸形和癫痫的重要原因，包括结节性硬化症 复杂的局灶性皮质发育不良、羊水过多、巨脑和症状性癫痫(PMSE) 综合征，与严重癫痫和智力残疾有关，由- 在STRADA基因中起作用。用雷帕霉素或其类似物抑制mTOR通路有助于 多发性疾病，但许多人仍然有癫痫发作，没有反应。先前的研究产生了干细胞 从PMSE患者身上分离出来，并在二维培养中将其分化为兴奋性神经元并进行背侧传代 人脑器官，类似于发育中的皮质的三维神经结构，主要是 含有兴奋性神经元。PMSE类器官表现为mTOR过度活动，体积增大，异常 形态、神经元分化延迟，以及外径向神经胶质细胞的增加，这是一种 对人类大脑的扩大负有责任。这些发现解释了PMSE中的巨脑症，但 STRADA功能丧失导致这些观察到的表型的中间机制不是 为人所知。此外，STRADA功能丧失对起源于 腹侧前脑结构尚不清楚。异常的抑制性中间神经元是癫痫的主要原因， 自闭症，都是多发性疾病的显著特征。这一提议将检验核心假设，即损失 STRADA通过mTOR复合体1过度活动而导致皮质畸形，而抑制性中间神经元 发育也受到损害，导致癫痫。这项创新的提案将使用背部命中的有机化合物。 为了确定Strada下游的哪些信号通路改变导致皮质发育改变， 并将确定代偿反馈信号是否会阻碍雷帕霉素治疗的有效性 (目标1)。我们还将产生腹型和背型有机化合物之间的融合以进行研究 神经元间发育、神经元间迁移、细胞类型特异性转录改变(目标2)以及 可导致癫痫的神经网络过度兴奋(目标3)。拟议的研究将具有重要的意义 影响，因为了解了PMSE发病机制的信号通路和细胞类型 将提供一个开发机械驱动疗法的平台，可以阻止或逆转癫痫的发生 我们的发现应该在更广泛的背景下适用于癫痫。

PROJECT SUMMARY Epilepsy is a major cause of morbidity, mortality, disability, and expense, and affects over 470,000 children in the U.S. While many medications to control seizures have been developed, about 30% of patients do not respond to medications, and to date, there are no medications that can prevent or halt the progression of epilepsy. Recently, many genetic causes of epilepsy have been identified, providing insights into pathways involved in epileptogenesis. Mutations causing hyperactivity of the mTOR pathway (so-called “mTORpathies”) have emerged as an important cause of cerebral malformations and epilepsy, including tuberous sclerosis complex, focal cortical dysplasia, and polyhydramnios, megalencephaly, and symptomatic epilepsy (PMSE) syndrome, which is associated with severe epilepsy and intellectual disability and is caused by a loss-of- function in the STRADA gene. Inhibition of the mTOR pathway with rapamycin or its analogs can help in mTORopathies, but many individuals still have seizures that do not respond. Prior studies generated stem cells from individuals with PMSE and differentiated them into excitatory neurons in 2-D culture and dorsally-fated human cerebral organoids, 3-D neural structures that resemble the developing cortex and predominantly contain excitatory neurons. The PMSE organoids demonstrated mTOR hyperactivity, increased size, abnormal morphology, delayed neuronal differentiation, and an increase in outer radial glia, a progenitor cell that is responsible for the expanded size of the human brain. These findings explain megalencephaly in PMSE, but the intermediate mechanisms by which STRADA loss-of-function results in these observed phenotypes is not known. Furthermore, the effect of STRADA loss-of-function on inhibitory interneurons that originate from ventral forebrain structures is not known. Abnormal inhibitory interneurons are a major cause of epilepsy and autism, both prominent features of mTORopathies. This proposal will test the central hypothesis that loss of STRADA causes cortical malformation by mTOR complex 1 hyperactivity, and that inhibitory interneuron development is also impaired, resulting in epilepsy. This innovative proposal will use dorsally-fated organoids to identify which signaling pathway alterations downstream of STRADA result in altered cortical development, and will determine whether compensatory feedback signals hinder the effectiveness of rapamycin treatment (Aim 1). We will also generate fusions between ventrally-fated and dorsally-fated organoids to study interneuron development, interneuron migration, cell-type specific transcriptional alterations (Aim 2), as well as neuronal network hyperexcitability that can result in epilepsy (Aim 3). The proposed study will have significant impact because understanding the signaling pathways and cell types responsible for pathogenesis in PMSE will provide a platform to develop mechanistically driven therapies that can halt or reverse epileptogenesis for mTORopathies, and our findings should be applicable to epilepsies in a broader context.