Single Nucleus Transcriptional Profiling of Intractable Focal Epilepsy

项目摘要 高达30%的癫痫患者有顽固性癫痫发作。目前尚不清楚癫痫发作的机制 在难治性局灶性癫痫中，发作和传播是区域性的，其中组织受累 异质性和区域性在癫痫的启动、传播和抵抗扩散中起着不同的作用。更好 需要了解癫痫发作焦点与半影区的生物学基础，在半影区是兴奋的 脑电出现放电，但神经元的反应受到完整抑制的限制。抑制性与非平衡性 兴奋性神经元输入(I/E失衡)是癫痫发生和传播的主要因素， 尤其是神经元间功能障碍和神经元/锥体细胞之间的相互作用。I/E失衡影响癫痫发作 在广泛的癫痫病理中发生；I/E失衡和神经元间功能的研究可以 从而揭示了许多类型和病因癫痫的共同途径。我们假设特定的细胞类型 改变导致在难治性局灶性癫痫手术中切除的大脑I/E失衡，而这些 转录改变将与组织病理学和局部脑电有关。我们将通过执行Single来测试这一点 核糖核酸测序(SnRNAseq)在MRI引导电生理定位活检中的应用 正在接受癫痫手术。这将提供细胞类型特定转录谱的特定位置关联 有组织学、放射学和电生理改变。我们将对癫痫发作的焦点进行采样 半影区，以确定细胞组成和细胞类型特定表达谱的差异，以及 确定癫痫发作发生和传播过程中涉及的改变。尽管我们将重点放在中间神经元 分析和I/E失衡，SNRNAseq将提供关于细胞类型多样性的数据。在AIM 1中，我们将使用 用SnRNAseq方法研究人脑切除后癫痫灶与半影区基因的差异表达 在难治性局灶性癫痫手术中，关注中间神经元与兴奋性锥体细胞转录 改装。微电极记录将提供癫痫发作的空间轨迹并定义焦点和 半影区。我们将通过比较不同区域和细胞类型的转录图谱来确定特定区域和细胞类型的变化 具有脑电特征的样本。因此，我们将确定癫痫发作中差异表达的分子和途径。 焦点与半暗带，突出可能是癫痫发作产生的主要过程。在AIM 2中，我们将表演 AIM 1中确定的顶级靶分子以及以前的分子的免疫组织化学分析 已确定在癫痫和癫痫发作的I/E失衡中发挥作用。因此，我们的研究将提供 磁共振引导电生理内细胞和亚细胞表达模式的显微分辨 局部活组织检查。阐明难治性局灶性癫痫发作的局部病理生理学可以提供 治疗的目标。

PROJECT ABSTRACT Up to 30% of individuals with epilepsy have intractable seizures. It is unclear how mechanisms underlying seizure onset and propagation operate regionally in intractable focal epilepsy, in which tissue involvement is heterogeneous and regions play different roles in seizure initiation, propagation and resistance to spread. Better understanding is needed of the biology underlying the seizure focus versus the penumbra, where excitatory discharges appear on EEG but neuronal response is limited by intact inhibition. Imbalance of inhibitory and excitatory neuronal inputs (I/E imbalance) is a primary factor underlying seizure generation and propagation, particularly interneuron dysfunction and interneuron/pyramidal cell interaction. I/E imbalance influences seizure generation in a wide array of epilepsy pathologies; investigation of I/E imbalance and interneuron function can thereby reveal pathways common to many types and causes of epilepsy. We hypothesize that cell-type specific alterations result in I/E imbalance in brain resected during surgery for intractable focal epilepsy, and that these transcriptional changes will correlate with histopathology and regional EEG. We will test this by performing single nucleus RNA sequencing (snRNAseq) on MRI-guided electrophysiologically-localized biopsies from patients undergoing epilepsy surgery. This will provide site-specific correlation of cell type-specific transcriptomic profiles with histological, radiographic, and electrophysiologic alterations. We will sample the seizure focus and penumbra to determine the differences in cellular composition and cell type-specific expression profiles, and identify alterations involved in seizure generation and propagation. Although we will focus on interneuron profiling and I/E imbalance, snRNAseq will provide data on the diversity of cell types. In AIM 1 we will use snRNAseq to identify differential expression of genes in seizure focus vs. penumbra in human brain resected during surgery for intractable focal epilepsy, focusing on interneuron vs excitatory pyramidal cell transcriptional alterations. Microelectrode recordings will provide the spatial trajectory of a seizure and define focus and penumbra. We will identify region- and cell type-specific alterations by comparing transcriptional profiles across EEG-characterized samples. We will thereby identify molecules and pathways differentially expressed in seizure focus vs. penumbra, highlighting processes likely to be primary to seizure generation. In AIM 2 we will perform immunohistochemical analyses of top target molecules identified in Aim 1, as well as molecules previously identified to play a role in I/E imbalance in epilepsy and seizure generation. Our studies will thereby provide microscopic resolution of cellular and subcellular patterns of expression within MRI-guided electrophysiologically localized biopsies. Illuminating the regional pathophysiology of seizures in intractable focal epilepsy can provide targets for therapy.