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

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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.