Single-cell transcriptome sequencing to investigate mechanisms of epileptogenesis in genetic mouse models and human brain biopsy tissue

尽管越来越多的癫痫基因被鉴定出来，并在简单的表达系统中阐明了致病突变的分子机制，但作为遗传缺陷的结果，癫痫的确切机制仍然知之甚少。具体地说，在遗传性癫痫综合征的癫痫表型的发展过程中，基因突变和癫痫发生是如何相互联系的，以及癫痫发作开始的时间点仍然难以捉摸。因此，使用最先进的测序技术，我们打算在我们的DFG资助的研究单位2715(RU为2715)的四个单独项目中，研究条件性敲入小鼠模型(Scn1a)和全球敲入癫痫小鼠模型(SCN2A，KCNA2)的不同神经元亚群中特定区域和时间特定的RNA表达。癫痫发生的转录标志将通过海马神经元(CA1和CA3)、皮质(S1)和丘脑(NRT)神经元的单细胞RNA测序来识别。此外，我们还将通过对人类海马区(波恩大学)和大脑皮层(Tübingen大学)活检组织中来自全身性遗传性癫痫(GGE)风险位点的候选SNPs和与癫痫发生有关的候选基因进行表观基因组分析，剖析导致全身性遗传性癫痫(GGE)风险增加的调节性SNPs(RSNPs)。这些工作将在已经为2715个项目建立的RU的框架内进行，以及来自Tübingen、Köln和波恩的PI与已经建立的科隆基因组学中心(CCG)的P.Nürnberg教授密切合作。与癫痫发生相关的单细胞测序产生的数据不仅将用于进一步加深我们对癫痫综合征发育贡献的理解，还将用于指导其人类遗传项目，以识别导致癫痫的新基因。

Despite the identification of a steadily increasing number of epilepsy genes and elucidation of molecular mechanisms of disease-causing mutations in simple expression systems, the exact mechanisms as to how epilepsy develops as a consequence of a genetic defect is poorly understood. Specifically, it remains elusive how genetic mutations and epileptogenesis are interconnected in the development of an epileptic phenotype in genetic epilepsy syndromes as well as at which time-points epileptic seizures start. Using state of the art sequencing techniques we, therefore, intend to investigate in four separate projects of our DFG-funded research unit 2715 (RU FOR 2715) brain region- and time-specific RNA expression in distinct neuronal subpopulations of a conditional knock-in mouse model (Scn1a) and global knock-in epilepsy mouse models (Scn2a, Kcna2). Transcriptomic signatures of epileptogenesis will be identified by single-cell RNA sequencing of hippocampal (CA1 and CA3), cortical (S1), and thalamic (nRT) neurons. In addition, we will dissect regulatory SNPs (rSNPs) conferring increased risk of generalized genetic epilepsies (GGE) by epigenomic profiling of candidate SNPs derived from GWAS risk loci of GGE and candidate genes implicated in epileptogenesis in human hippocampal (University of Bonn) and cortical (University of Tübingen) biopsies. The efforts will be performed within the framework of the already established RU FOR 2715 projects and a close collaboration of the PIs from Tübingen, Köln and Bonn with Prof. P. Nürnberg, Cologne Center for Genomics (CCG), which is already established. The generated data of the single cell sequencing related to epileptogenesis will not only be used to further deepen our understanding of the developmental contribution in epilepsy syndromes, but also utilized in the Research unit to guide its human genetic projects to identify new genes causing epilepsy.