Molecular and cellular basis of Lessel-Kreienkamp syndrome, caused by pathogenic variants in AGO2

由 AGO2 致病性变异引起的 Lessel-Kreienkamp 综合征的分子和细胞基础

• 批准号: 514647696
• 负责人: Professor Dr. Hans-Jürgen Kreienkamp
• 金额: --
• 依托单位: Institut für Humangenetik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/514647696?language=en
• 关键词: Molecular; cellular; basis; Lessel; Kreienkamp

RNA interference is the major mechanism for post-transcriptional regulation of gene expression in eukaryotic cells. Precursors of microRNAs (miRNAs) are transcribed, processed into mature miRNAs and loaded onto Argonaute (AGO1-4) proteins to form the RNA-induced silencing complex (RISC). Each miRNA recognizes target mRNAs by base pairing, leading to translational silencing and mRNA degradation in cytoplasmic processing (P-) bodies. We have recently linked pathogenic variants in AGO2 to a neurodevelopmental disorder characterized by intellectual disability, delayed motor development, impaired speech and receptive language development. This disorder has now been named Lessel-Kreienkamp syndrome (LESKRES). Missense variants in AGO2 reduce the capacity of the encoded protein to perform shRNA based silencing in in vitro assays. Intriguingly, comparable variants in similarly affected individuals were later also identified in AGO1. The impact of the variants at the molecular, cellular and clinical level remains unclear. So far, there are no cellular or animal models for this disorder. Thus, we do not know (i) how genotype correlates with phenotype; (ii) which function(s) of AGO2 are affected; (iii) whether the complement of neuronal miRNAs is altered; and (iv) which targets of the AGO2/miRNA complex are dysregulated in neuronal systems. Here we will address these questions by using several complementary approaches. By combining clinical details with in-vitro analyses, we will investigate variant-specific effects on the clinical outcome of LESKRES disorder. We will analyse the impact of missense variants on non-canonical AGO2 functions, such as the regulation of alternative splicing and the DNA damage response pathway. Moreover, we will determine how pathogenic amino acid exchanges in AGO2 as well as AGO1 alter the set of RISC-associated miRNAs in murine neurons, and in iNeurons differentiated from induced pluripotent stem (iPS) cells of individuals harbouring missense variants in AGO2. Besides a detailed analysis of the effects of AGO2 variants on gene expression, we will evaluate cultured neurons for changes in morphology, synapse formation, and alterations in signalling pathways. In a further approach, we have generated two mouse lines carrying missense variants, identified in LESKRES-individuals, and one loss-of-function line. In brains of these mice, we will determine the effect of Ago2 variants on the complement of miRNAs and their mRNA targets. We will assess how these changes affect the cellular and the synaptic proteome, as well as synaptic function and plasticity. Finally, we will analyse the behaviour of mice with respect to changes in learning and memory paradigms. We expect to obtain a clearer view on alterations in gene expression occurring due to variants in AGO2 found in affected individuals. In addition, we expect to create models of the human disease that will provide avenues for the exploration of therapeutic approaches.

RNA干扰是真核细胞中基因表达转录后调控的主要机制。 microRNA (miRNA) 的前体被转录、加工成成熟的 miRNA 并加载到 Argonaute (AGO1-4) 蛋白上，形成 RNA 诱导的沉默复合物 (RISC)。每个 miRNA 通过碱基配对识别目标 mRNA，导致细胞质加工 (P-) 体中的翻译沉默和 mRNA 降解。我们最近将 AGO2 的致病变异与一种神经发育障碍联系起来，其特征是智力障碍、运动发育迟缓、言语和接受性语言发育受损。这种疾病现在被命名为 Lessel-Kreienkamp 综合征 (LESKRES)。 AGO2 中的错义变异降低了编码蛋白在体外测定中执行基于 shRNA 的沉默的能力。有趣的是，后来在 AGO1 中也发现了类似受影响个体的类似变异。这些变异在分子、细胞和临床水平上的影响仍不清楚。到目前为止，还没有这种疾病的细胞或动物模型。因此，我们不知道（i）基因型与表型如何相关； (ii) AGO2 的哪些功能受到影响； (iii) 神经元 miRNA 的补体是否发生改变； (iv) AGO2/miRNA 复合物的哪些靶标在神经系统中失调。在这里，我们将通过使用几种互补的方法来解决这些问题。通过将临床细节与体外分析相结合，我们将研究变异特异性对 Leskres 疾病临床结果的影响。我们将分析错义变异对非规范 AGO2 功能的影响，例如选择性剪接的调节和 DNA 损伤反应途径。此外，我们将确定 AGO2 和 AGO1 中的致病性氨基酸交换如何改变小鼠神经元中的 RISC 相关 miRNA 集，以及从含有 AGO2 错义变异的个体的诱导多能干 (iPS) 细胞分化而来的 iNeurons 中。除了详细分析 AGO2 变体对基因表达的影响外，我们还将评估培养的神经元的形态变化、突触形成和信号通路的改变。在进一步的方法中，我们生成了两个携带错义变体的小鼠品系（在 LESKRES-个体中鉴定）和一个功能丧失品系。在这些小鼠的大脑中，我们将确定 Ago2 变体对 miRNA 及其 mRNA 靶标的补充的影响。我们将评估这些变化如何影响细胞和突触蛋白质组，以及突触功能和可塑性。最后，我们将分析小鼠在学习和记忆范式变化方面的行为。我们希望能够更清楚地了解受影响个体中发现的 AGO2 变异导致的基因表达变化。此外，我们期望创建人类疾病模型，为探索治疗方法提供途径。