Modelling of nonmuscle actinopathies in brain organoids

• Applicant: Professorin Dr. Nataliya Di Donato
• Subject Area: Molecular and Cellular Neurology and Neuropathology ; Human Genetics
• Term: from 2020 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 433057035

This project focuses on assessment of neuronal differentiation under the presence of beta- and gamma-actin mutations. We apply for performing a droplet-based single cell RNA sequencing to compare cell diversity in brain organoids derived from patients with mutations in ACTB and ACTG1 with control and CRISPR/Cas edited cell lines. ACTB and ACTG1 encode for two nonmuscle actin isoforms that represent the main actin isoforms in the neuronal cells. On the protein level these two isoforms differ by only four N-terminal amino acids but undergo different posttranslational modifications and have specific and non-redundant functions. In fibroblastic, epithelial and endothelial cells gamma-actin enriches in sub-membranous networks and is associated with cell migration; beta-actin is implicated in contractile processes and accordingly localizes to stress fibers and cell-cell contacts. However, studies addressing actin isoform expression in the neuronal tissue are conflicting and no data is available regarding actin isoform specific functions in the neuronal cells.Heterozygous mutations in ACTB and ACTG1 are associated with a group of Mendelian disorders encompassed under a collective term nonmuscle actinopathies (NMAs). Taking into account the various roles of actin cytoskeleton in the brain cells, the primary association of NMAs with intellectual disability is self-evident. Structural malformation - lissencephaly – is a part of the NMAs clinical spectrum. Lissencephaly is defined as a malformation of cortical development caused by insufficient neuronal migration. However, our preliminary experiments on patient derived brain organoids with ACTB and ACTG1 mutations clearly demonstrated abnormal formation of the neuroectoderm indicating that actin mutations interrupt multiple developmental steps and not only neuronal migration. NMAs phenotype is variable even in patients with the same mutation and cortical malformation is present in some but not all patients. The set of our experiments is designed to model the influence of extrinsic (e.g. hypoxia) modifiers on development of organoids with the ACTB hot-spot mutation with the further assessment of cell diversity applying single cell RNA-sequencing.By the end of the project we expect to gain insights into actin-dependent regulation of neuronal differentiation. Additionally we anticipate revealing the role of prenatal hazards in clinical variability of NMAs and actin function during neuronal development.

• DFG Programme: Research Grants