Elucidating pathogenic mechanisms in STRADA-related brain malformation and epilepsy

项目摘要 癫痫是发病率、死亡率、残疾和费用的主要原因，并且影响美国超过470，000名儿童。 虽然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，还没有药物可以阻止或阻止 癫痫最近，许多癫痫的遗传原因已被确定，提供了对途径的见解 与癫痫发生有关导致mTOR通路过度活跃的突变（所谓的“mTOR病”） 已经成为脑畸形和癫痫的重要原因，包括结节性硬化症 复杂、局灶性皮质发育不良、羊水过多、巨脑畸形和症状性癫痫（PMSE） 综合征，这是与严重的癫痫和智力残疾，是由损失- STRADA基因的功能。用雷帕霉素或其类似物抑制mTOR通路可以帮助抑制细胞凋亡。 mTORopathies，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个体，并在二维培养中将其分化为兴奋性神经元， 人类大脑类器官，类似于发育中的皮质的3D神经结构， 含有兴奋性神经元PMSE类器官表现出mTOR过度活跃，大小增加，异常 形态学，延迟的神经元分化，以及外放射状胶质细胞的增加，外放射状胶质细胞是一种祖细胞， 是人类大脑体积扩大的原因这些发现解释了PMSE中的巨脑畸形， STRADA功能丧失导致这些观察到的表型的中间机制不是 知道的此外，STRADA功能丧失对抑制性中间神经元的影响， 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，这两种mTORopathies的突出特征。这一提议将检验中心假设， STRADA通过mTOR复合物1过度活跃引起皮质畸形， 发育也受到损害，导致癫痫。这项创新的提案将使用背缘类器官 为了确定STRADA下游的信号通路改变导致皮质发育改变， 并将确定补偿反馈信号是否阻碍雷帕霉素治疗的有效性 (Aim 1）。我们还将在腹侧命运和背侧命运的类器官之间产生融合， 中间神经元发育、中间神经元迁移、细胞类型特异性转录改变（Aim 2），以及 神经元网络过度兴奋，可导致癫痫（目的3）。这项研究将具有重大意义。 因为了解PMSE发病机制的信号通路和细胞类型 将提供一个平台，开发机械驱动的治疗，可以停止或逆转癫痫发生， mTORopathies，我们的研究结果应该适用于癫痫在更广泛的背景下。

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.