Multidimensional Functional Profiling of Human Neuropathogenic FOXG1 Alleles in Primary Cultures of Murine Pallial Precursors

FOXG1 is an ancient transcription factor gene mastering telencephalic development. Several structural FOXG1 mutations lead to the “FOXG1 syndrome”, a complex and heterogeneous neuropathological entity, for which no cure is presently available. Reconstruction of primary neurodevelopmental/physiological anomalies evoked by these mutations is an obvious pre-requisite for future, precision therapy of such syndrome. As a proof-of-principle, we functionally scored three FOXG1 neuropathogenic alleles, FOXG1G224S, FOXG1W308X, and FOXG1N232S, against their healthy counterpart. Specifically, we delivered transgenes encoding for them to dedicated preparations of murine pallial precursors and quantified their impact on selected neurodevelopmental and physiological processes mastered by Foxg1: pallial stem cell fate choice, proliferation of neural committed progenitors, neuronal architecture, neuronal activity, and their molecular correlates. Briefly, we found that FOXG1G224S and FOXG1W308X generally performed as a gain- and a loss-offunction- allele, respectively, while FOXG1N232S acted as a mild loss-of-function allele or phenocopied FOXG1WT. These results provide valuable hints about processes misregulated in patients heterozygous for these mutations. Moreover, they suggest that murine pallial cultures may be employed for fast multidimensional profiling of novel, human neuropathogenic FOXG1 alleles. Next, as a further step towards precision therapy of such syndrome, we assessed the ability of small RNAs activating Foxg1 transcription or destabilizing its mRNA (by RNAa and RNAi, respectively) to impact expression levels of selected Foxg1 targets and modulate electrical activity of neocortical cultures. Moreover, we preliminarily evaluated their performances in cultures of striatal precursors.