Thalamocortical network dysfunction in a novel genetic model of GRIN2D developmental and epileptic encephalopathy

项目总结 发育和癫痫脑病(DeE)是一组严重的儿童癫痫发作障碍， 一个重要的和多样化的遗传成分。DeE患者通常会经历严重的癫痫负担和 患有认知和发育障碍，以及睡眠和运动障碍。50或 更多含有导致DIE的突变的基因包括“GRIN”基因，它编码 膜蛋白复合体对于神经元之间的电化学信号传递是重要的。基因突变 GRIN2D亚基引起特别严重和难治性的DIE。我们最近开发了一种小鼠模型 GRIN2D携带一种突变，在GRIN2D Dee患者中多次复发。这些老鼠有非常多的 强健的癫痫特征，包括已知的惊厥性发作和非惊厥性发作 由大脑皮层和丘脑(即丘脑皮质网络)之间的信号调节。这 丘脑皮质网络也与睡眠、意识和许多其他活动的调节有关 大脑区域。在初步发现中，我们还注意到Grin2D的蛋白质产物在 大脑皮质的兴奋性和抑制性细胞，而丘脑的抑制性细胞则丰富。 此外，神经元信号传递的主要部位--突触--在 大脑皮层，表明它们不能正常发育。总而言之，这些初步数据激励着 研究Grin2D基因突变如何改变大脑皮质和丘脑的功能。在这个试点项目中，我们 将应用电生理学方法测量GRIN2D Dee模型中突触的功能 确定结构变化是否与功能损伤相对应。我们还将识别神经细胞 在GRIN2D Dee的丘脑皮质网络内受影响最严重的连接。Aim1将使用 检查大脑皮质和丘脑内群体水平反应的切片电生理学方法 对于这些区域产生癫痫样活动的易感性。我们还将采用创新的方法来 解剖大脑皮质反应的组成部分，这将使我们能够有效地定位 大脑皮层中的许多连接，很可能是由Grin2D突变改变的。这些网络级别 将使用突触的细胞内全细胞膜片钳记录进一步探讨AIM2的作用 洋流。AIM2将评估两种不同的GRIN2D信号模式，突触和强直，这两种模式是相关的 具有不同的细胞后果，可能参与了GRIN2D Dee的致病机制。 这些实验将共同确定GRIN2D Dee突变对酶活性的影响 神经回路，与这些动物癫痫的产生有关，并将在 细胞水平，癫痫发生的潜在机制。这项研究是确定 GRIN2D如何影响丘脑皮质回路的发育和维持(在正常情况下 以及在DIE模型中)，这些信息将指导未来开发有针对性的矫正疗法的努力。

PROJECT SUMMARY Developmental and epileptic encephalopathy (DEE) is a collection of severe childhood seizure disorders, with a significant and diverse genetic component. DEE patients usually experience a significant seizure burden and suffer from cognitive and developmental impairments, as well as sleep and motor disturbances. The 50 or more genes harboring DEE-causing mutations include the “GRIN” genes, which encode components of membrane protein complexes that are important for electrochemical signaling between neurons. Mutations in the GRIN2D subunit cause particularly severe and intractable DEE. We recently developed a mouse model of GRIN2D carrying a mutation that has recurred several times in GRIN2D DEE patients. These mice have very robust epileptic features, including both convulsive seizures and non-convulsive seizures known to be regulated by signaling between the cerebral cortex and the thalamus (i.e. thalamocortical network). This thalamocortical network is also associated with regulation of sleep, awareness, and the activity of many other brain regions. In preliminary findings, we also noticed that the protein product of Grin2D is expressed in excitatory and inhibitory cells of the cortex, while it is enriched in the inhibitory cells of the thalamus. Additionally, the primary sites of neuronal signaling – the synapse – have unusual structural features in the cortex, suggesting that they do not develop normally. Altogether, these preliminary data motivate an examination of how mutation in Grin2D alters the function of the cortex and thalamus. In this pilot program, we will apply electrophysiological approaches to measure the function of synapses in the GRIN2D DEE model to determine if structural changes correspond to functional impairments. We will also identify the neuronal connections that are most severely affected within the thalamocortical network in GRIN2D DEE. Aim1 will use slice electrophysiology methods to examine population-level responses within the cortex and thalamus to look for susceptibility of these regions to generate seizure-like activity. We will also adapt innovative approaches to dissect the component parts of the response in the cortex, which will allow us to efficiently pinpoint amongst the many connections in the cortex, which are most likely altered by the Grin2D mutation. These network-level effects will be further explored in Aim2 using intracellular whole-cell patch clamp recordings of synaptic currents. Aim2 will assess two distinct modes of GRIN2D signaling, synaptic and tonic, which are associated with different cellular consequences and are likely involved in the GRIN2D DEE pathogenic mechanism. Together these experiments will determine the consequence of the GRIN2D DEE mutation on the activity of neural circuits, which are implicated in the generation of seizures in these animals, and will determine, at the cellular level, the underlying mechanism of epileptogenesis. This study is an important first step for determining how GRIN2D shapes the development and maintenance of thalamocortical circuits (under normal conditions and in a DEE model), information which will guide future efforts to develop targeted corrective therapeutics.