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干扰是真核细胞转录后基因表达调控的主要机制。microrna的前体（mirna）被转录、加工成成熟的mirna并装载到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-）体的翻译沉默和mRNA降解。我们最近将AGO2的致病变异与一种以智力残疾、运动发育迟缓、语言和接受性语言发育受损为特征的神经发育障碍联系起来。这种疾病现在被命名为Lessel-Kreienkamp综合征（LESKRES）。在体外实验中，AGO2中的错义变异体降低了编码蛋白执行shRNA沉默的能力。有趣的是，在类似的个体中，类似的变异后来也在AGO1中被发现。这些变异在分子、细胞和临床水平上的影响尚不清楚。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(1)基因型如何与表型相关；（ii） AGO2的哪些功能受到影响；（iii）神经元mirna的补体是否改变；（iv）在神经元系统中，AGO2/miRNA复合物的哪些靶点失调。在这里，我们将使用几种互补的方法来解决这些问题。通过结合临床细节和体外分析，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，如调节选择性剪接和DNA损伤反应途径。此外，我们将确定AGO2和AGO1中致病性氨基酸交换如何改变小鼠神经元中risc相关的mirna，以及从AGO2中携带错义变体的诱导多能干细胞（iPS）分化的神经元中。除了详细分析AGO2变异对基因表达的影响外，我们还将评估培养神经元在形态学、突触形成和信号通路改变方面的变化。在进一步的方法中，我们产生了两个携带错义变体的小鼠系，在leskres -个体中鉴定，以及一个功能丧失系。在这些小鼠的大脑中，我们将确定Ago2变异对mirna补体及其mRNA靶标的影响。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。我们希望对受影响个体中AGO2变异引起的基因表达改变有更清晰的认识。此外，我们期望创建人类疾病的模型，为探索治疗方法提供途径。