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

Abstract

FOXG1 is an ancient transcription factor gene mastering telencephalic development. A number of distinct 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. Here, 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-of-function-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, to be re-addressed more stringently in patient iPSC-derivative neuro-organoids. Moreover, they suggest that murine pallial cultures may be employed for fast multidimensional profiling of novel, human neuropathogenic FOXG1 alleles, namely a step propedeutic to timely delivery of therapeutic precision treatments.

Dual role of FOXG1 in regulating gliogenesis in the developing neocortex via the FGF signaling pathway

“…Foxg1 was previously identified as a regulator of sequential neurogenesis in the neocortex via controlling the timing and order of generation of specific neuronal subtypes (26, 29, 30, 40). Foxg1 overexpression in E12 cortical progenitors in vitro was shown to suppress gliogenesis (32, 33). However, in these studies, the Fgf pathway was not identified as a possible FOXG1 target, and Foxg1 knockdown did not result in gliogenesis.…”

“…However, in these studies, the Fgf pathway was not identified as a possible FOXG1 target, and Foxg1 knockdown did not result in gliogenesis. Therefore, the endogenous role of FOXG1 could not be established (32, 33).…”

“…Forkhead family transcription factor FOXG1 is a well-established regulator of the sequential production of neuronal subtypes in the mammalian neocortex (25–30). Further support for a neurogenic role for this factor came from overexpression experiments in vitro or cultured progenitors transplanted in vivo , in which decreased gliogenesis and downregulation of progliogenic pathways and markers were seen (31–33). However, in these studies, Foxg1 knockdown did not cause significant gliogenesis (31, 32).…”

Foxg1 regulates translation of neocortical neuronal genes, including the main NMDA receptor subunit gene,Grin1

“…Later, it promotes morphological maturation of glutamatergic (Brancaccio et al, 2010; Chiola et al, 2019; Yu et al, 2019) and gabaergic (Zhu et al, 2019) telencephalic neurons. Moreover, Foxg1 sustains activity and excitability of these neurons (Frisari et al, 2022; Tigani et al, 2020; Zhu et al, 2019), via an articulated impact on transcription of specific gene-sets (Artimagnella and Mallamaci, 2019; Tigani et al, 2020). Besides, it is in turn transiently promoted by neuronal activity (Fimiani et al, 2016; Tigani et al, 2020).…”

“…Foxg1 is an ancient transcription factor mastering a number of developmental processes that take place in the rostral brain. These include early activation of pan- telencephalic (Hanashima et al, 2007), subpallial (Manuel et al, 2010), and paleo-neo- pallial (Muzio and Mallamaci, 2005) programs, promotion of neural precursors selfrenewal (Martynoga et al, 2005), balance between neuronogenesis and gliogenesis (Brancaccio et al, 2010; Falcone et al, 2019; Frisari et al, 2022; Santo et al, 2022), and laminar specification of neocortical neurons (Hanashima et al, 2004; Miyoshi and Fishell, 2012; Toma et al, 2014). Later, it promotes morphological maturation of glutamatergic (Brancaccio et al, 2010; Chiola et al, 2019; Yu et al, 2019) and gabaergic (Zhu et al, 2019) telencephalic neurons.…”