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. 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.

The dual action of glioma-derived exosomes on neuronal activity: synchronization and disruption of synchrony.

Seizures represent a frequent symptom in gliomas and significantly impact patient morbidity and quality of life. Although the pathogenesis of tumor-related seizures is not fully understood, accumulating evidence indicates a key role of the peritumoral microenvironment. Brain cancer cells interact with neurons by forming synapses with them and by releasing exosomes, cytokines, and other small molecules. Strong interactions among neurons often lead to the synchronization of their activity. In this paper, we used an in vitro model to investigate the role of exosomes released by glioma cell lines and by patient-derived glioma stem cells (GSCs). The addition of exosomes released by U87 glioma cells to neuronal cultures at day in vitro (DIV) 4, when neurons are not yet synchronous, induces synchronization. At DIV 7-12 neurons become highly synchronous, and the addition of the same exosomes disrupts synchrony. By combining Ca2+ imaging, electrical recordings from single neurons with patch-clamp electrodes, substrate-integrated microelectrode arrays, and immunohistochemistry, we show that synchronization and de-synchronization are caused by the combined effect of (i) the formation of new neuronal branches, associated with a higher expression of Arp3, (ii) the modification of synaptic efficiency, and (iii) a direct action of exosomes on the electrical properties of neurons, more evident at DIV 7-12 when the threshold for spike initiation is significantly reduced. At DIV 7-12 exosomes also selectively boost glutamatergic signaling by increasing the number of excitatory synapses. Remarkably, de-synchronization was also observed with exosomes released by glioma-associated stem cells (GASCs) from patients with low-grade glioma but not from patients with high-grade glioma, where a more variable outcome was observed. These results show that exosomes released from glioma modify the electrical properties of neuronal networks and that de-synchronization caused by exosomes from low-grade glioma can contribute to the neurological pathologies of patients with brain cancers.

Foetal neural progenitors contribute to postnatal circuits formation ex vivo: an electrophysiological investigation.

Neuronal progenitor cells (NPC) play an essential role in homeostasis of the central nervous system (CNS). Considering their ability to differentiate into specific lineages, their manipulation and control could have a major therapeutic impact for those CNS injuries or degenerative diseases characterized by neuronal cell loss. In this work, we established an in vitro co-culture and tested the ability of foetal NPC (fNPC) to integrate among post-mitotic hippocampal neurons and contribute to the electrical activity of the resulting networks. We performed extracellular electrophysiological recordings of the activity of neuronal networks and compared the properties of spontaneous spiking in hippocampal control cultures (HCC), fNPC, and mixed circuitries ex vivo. We further employed patch-clamp intracellular recordings to examine single-cell excitability. We report of the capability of fNPC to mature when combined to hippocampal neurons, shaping the profile of network activity, a result suggestive of newly formed connectivity ex vivo.