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

单细胞转录组测序研究遗传小鼠模型和人脑活检组织中癫痫发生的机制

• 批准号: 433112721
• 负责人: Professor Dr. Albert Becker
• 金额: --
• 依托单位: Klinik für Neurologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2020
• 资助国家: 德国
• 起止时间: 2019-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/433112721?language=en
• 关键词: Single; cell; transcriptome; sequencing; investigate

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.

尽管癫痫基因的鉴定数量稳步增加，并阐明了简单表达系统中致病突变的分子机制，但对癫痫如何因遗传缺陷而发展的确切机制知之甚少。具体来说，它仍然是难以捉摸的基因突变和癫痫发生是如何相互联系的癫痫表型的遗传性癫痫综合征的发展，以及在哪个时间点癫痫发作开始。因此，使用最先进的测序技术，我们打算在DFG资助的研究单位2715（RU FOR 2715）的四个独立项目中研究条件性敲入小鼠模型（Scn1a）和整体敲入癫痫小鼠模型（Scn2a，Kcna 2）的不同神经元亚群中脑区域和时间特异性RNA表达。癫痫发生的转录组学特征将通过海马（CA1和CA3）、皮质（S1）和丘脑（nRT）神经元的单细胞RNA测序来鉴定。此外，我们将通过对来自GGE的GWAS风险基因座的候选SNP和人类海马（波恩大学）和皮质（蒂宾根大学）活检中涉及癫痫发生的候选基因进行表观基因组分析，解剖赋予全身性遗传性癫痫（GGE）风险增加的调节性SNP（rSNP）。这些工作将在已经建立的RU FOR 2715项目的框架内进行，并与来自图宾根、科隆和波恩的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，还将用于研究单位指导其人类遗传项目，以确定导致癫痫的新基因。