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

RNA干扰是真核细胞基因表达转录后调控的主要机制。MicroRNAs的前体(MiRNAs)被转录、加工成成熟的miRNAs并装载到ArgAerte(Ago1-4)蛋白上形成RNA诱导沉默复合体(RISC)。每个miRNA通过碱基配对识别目标mRNAs，导致翻译沉默和细胞质加工(P-)体的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在一起，这种神经发育障碍的特征是智力残疾、运动发育延迟、言语受损和接受性语言发育。这种疾病现在被命名为Lessel-Kreienkamp综合征(LESKRES)。在体外实验中，AGO2中的错义变体降低了编码蛋白执行基于shRNA的沉默的能力。有趣的是，后来在AGO1中也发现了类似的受影响个体的可比变异。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(I)基因型与表型的相关性；(Ii)agO2的哪些功能(S)受到影响；(Iii)神经元miRNAs的补体是否发生变化；以及(Iv)agO2/miRNA复合体的哪些靶点在神经系统中调节失调。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，我们将调查变异特异性对LESKRES疾病临床结果的影响。我们将分析错义变异对AGO2非规范功能的影响，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中的致病氨基酸交换如何改变小鼠神经元中RISC相关的miRNAs集，以及在含有AGO2错义变异的个体的诱导多能干细胞(IPS)分化的iNeurons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的形态变化、突触形成和信号通路的变化。在另一种方法中，我们已经产生了两个携带错义变体的小鼠品系，在LESKRES-个体中鉴定，以及一个功能丧失品系。在这些小鼠的大脑中，我们将确定Ago2变体对miRNAs及其信使核糖核酸靶标的影响。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。我们希望对在受影响的个体中发现的AGO2变异引起的基因表达变化有更清晰的看法。此外，我们希望创建人类疾病的模型，为探索治疗方法提供途径。

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.