Lessel-KreienkampSyndrom:Molekulare und zelluläre Grundlagen

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 may recognize a set of target mRNAs by base pairing, which leads to translational silencing and mRNA degradation in cytoplasmic structures referred to as processing (P-) bodies. We have recently linked pathogenic variants in the AGO2 to a novel neurodevelopmental disorder, which has in the meanwhile been named Lessel-Kreienkamp syndrome (LESKRES), and is characterized by intellectual disability, delayed motor development, impaired speech and receptive language development. Our initial functional characterization showed that missense variants in AGO2 lead to a reduced 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 the AGO1. However, the impact of the variants on the clinical, molecular and cellular level remains somewhat unclear, and there are currently no available cellular and animal models. In more detail, it remains to be elucidated (i) the genotype-phenotype correlation, (ii) which function(s) of AGO2 are affected, (iii) how is the complement of miRNAs altered, (iv) which targets of the AGO2/miRNA complex are dysregulated in the neuronal systems, and how this affects the proteome. Here we will address these questions by using several complementary approaches. By combining the clinical details with already established in-vitro analyses, we will investigate the putative clinical variant-specific effects of the LESKRES disorder. Additionally, 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 affected individuals harbouring specific 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 (collaboration partner at UKE Hamburg), identified in LESKRES-indviduals, and one loss-of-function line. In brain tissues 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. Furthermore, we will address how Ago2 variants affect synaptic function and plasticity. Finally, we will analyse the behaviour of mice with respect to changes in learning and memory paradigms. As a result, 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 neuronal and mouse models of the human disease that will provide avenues for the exploration of possible therapeutic approaches.