The Forkead Box G1 (FOXG1) gene encodes for a DNA−binding transcription factor, essential for the development of the telencephalon in mammalian forebrain. Mutations in FOXG1 have been reported to be involved in the onset of Rett Syndrome, for which sequence alterations of MECP2 and CDKL5 are known. While visual alterations are not classical hallmarks of Rett syndrome, an increasing body of evidence shows visual impairment in patients and in MeCP2 and CDKL5 animal models. Herein we focused on the functional role of FOXG1 in the visual system of animal models (Foxg1+/Cre mice) and of a cohort of patients carrying FOXG1 mutations or deletions. Visual physiology of Foxg1+/Cre mice was assessed by visually evoked potentials, which revealed a significant reduction in response amplitude and visual acuity with respect to wild-type littermates. Morphological investigation showed abnormalities in the organization of excitatory/inhibitory circuits in the visual cortex. No alterations were observed in retinal structure. By examining a cohort of FOXG1-mutated patients by means of a panel of neuro-ophthalmological evaluations, we found that all of them exhibited visual alterations compatible with high level visual dysfunctions. In conclusion our data show that Foxg1 haploinsufficiency results in an impairment of mouse and patient visual function. Response to Reviewers: Pisa February 27th 2016Dear Editor, We would like to thank the referees for the thoughtful critiques on our manuscript and for the opportunity to revise our work. Please find enclosed a revised version of our manuscript (NSC-15-1232) entitled "Visual impairment in FOXG1-mutated patients and mice" by Boggio et al. As requested, we performed additional experiments on retina and visual cortex in order to more quantitavely prove the structure specific alterations and strengthen our conclusions. We included below a detailed response to the reviewer comments (in italics). References have been updated and added as suggested by referees. Sincerely, Tommaso PizzorussoReviewer #1:Major concerns 1.The description of the morphology of the five classes of retinal neurons would benefit from a quantitative approach amenable to statistical comparisons, similar to that performed for cortical GABAergic neurons in Fig. 4.We performed the requested quantitative analysis that is now shown in fig. 4. The analysis was focussed on cells in the ganglion cell layer because this layer is known to be affected in Foxg1 null mice. No significant change in the overall number of cells, and in ganglion cells identified using the RBPMS cell type-specific antibody, was present in Foxg1 heterozygous mice 2.There are no explicit statements regarding blinding of investigators to mouse genotypes during data acquisition and analyses, the use of Power Analysis to determine sample sizes, and predetermined criteria to exclude data sets. These are all critical issues that need to be performed and explicitly reported as recommended (Landis et al. Nature 2012).We included in the methods the fol...

BackgroundGlioblastomas are largely unresponsive to all available treatments and there is therefore an urgent need for novel therapeutics. Here we have probed the antineoplastic effects of a bacterial protein toxin, the cytotoxic necrotizing factor 1 (CNF1), in the syngenic GL261 glioma cell model. CNF1 produces a long-lasting activation of Rho GTPases, with consequent blockade of cytodieresis in proliferating cells and promotion of neuron health and plasticity.MethodsWe have tested the antiproliferative effects of CNF1 on GL261 cells and human glioma cells obtained from surgical specimens. For the in vivo experiments, we injected GL261 cells into the adult mouse visual cortex, and five days later we administered either a single intracerebral dose of CNF1 or vehicle. To compare CNF1 with a canonical antitumoral drug, we infused temozolomide (TMZ) via minipumps for 1 week in an additional animal group.ResultsIn culture, CNF1 was very effective in blocking proliferation of GL261 cells, leading them to multinucleation, senescence and death within 15 days. CNF1 had a similar cytotoxic effect in primary human glioma cells. CNF1 also inhibited motility of GL261 cells in a scratch-wound migration assay. Low dose (2 nM) CNF1 and continuous TMZ infusion significantly prolonged animal survival (median survival 35 days vs. 28 days in vehicle controls). Remarkably, increasing CNF1 concentration to 80 nM resulted in a dramatic enhancement of survival with no obvious toxicity. Indeed, 57% of the CNF1-treated animals survived up to 60 days following GL261 glioma cell transplant.ConclusionsThe activation of Rho GTPases by CNF1 represents a novel potential therapeutic strategy for the treatment of central nervous system tumors.

Mutations in the coding sequence of the X-linked gene MeCP2 (Methyl CpG–binding protein) are present in around 80% of patients with Rett Syndrome, a common cause of intellectual disability in female and to date without any effective pharmacological treatment. A relevant, and so far unexplored feature of RTT patients, is a marked reduction in peripheral circulation. To investigate the relationship between loss of MeCP2 and this clinical aspect, we used the MeCP2 null mouse model B6.129SF1-MeCP2tm1Jae for functional and pharmacological studies. Functional experiments were performed on isolated resistance mesenteric vessels, mounted on a pressurized myograph. Vessels from female MeCP2+/− mice show a reduced endothelium-dependent relaxation, due to a reduced Nitric Oxide (NO) availability secondary to an increased Reactive Oxygen Species (ROS) generation. Such functional aspects are associated with an intravascular increase in superoxide anion production, and a decreased vascular eNOS expression. These alterations are reversed by curcumin administration (5% (w/w) dietary curcumin for 21 days), which restores endothelial NO availability, decreases intravascular ROS production and normalizes vascular eNOS gene expression. In conclusion our findings highlight alterations in the vascular/endothelial system in the absence of a correct function of MeCP2, and uncover related cellular/molecular mechanisms that are rescued by an anti-oxidant treatment.

Mutations in the Forkhead box G1 (FOXG1) gene, a brain specific transcriptional factor, are responsible for the congenital variant of Rett syndrome. Until now FOXG1 point mutations have been reported in 12 Rett patients. Recently seven additional patients have been reported with a quite homogeneous severe phenotype designated as the FOXG1 syndrome. Here we describe two unrelated patients with a de novo FOXG1 point mutation, p.Gln46X and p.Tyr400X, respectively, having a milder phenotype and sharing a distinctive facial appearance. Although FoxG1 action depends critically on its binding to chromatin, very little is known about the dynamics of this process. Using fluorescence recovery after photobleaching, we showed that most of the GFP-FoxG1 fusion protein associates reversibly to chromatin whereas the remaining fraction is bound irreversibly. Furthermore, we showed that the two pathologic derivatives of FoxG1 described in this paper present a dramatic alteration in chromatin affinity and irreversibly bound fraction in comparison with Ser323fsX325 mutant (associated with a severe phenotype) and wild type Foxg1 protein. Our observations suggest that alterations in the kinetics of FoxG1 binding to chromatin might contribute to the pathological effects of FOXG1 mutations.

A 2A adenosine receptors are considered an excellent target for drug development in several neurological and psychiatric disorders. It is noteworthy that the responses evoked by A 2A adenosine receptors are regulated by D 2 dopamine receptor ligands. These two receptors are coexpressed at the level of the basal ganglia and interact to form functional heterodimers. In this context, possible changes in A 2A adenosine receptor functional responses caused by the chronic blockade/activation of D 2 dopamine receptors should be considered to optimise the therapeutic effectiveness of dopaminergic agents and to reduce any possible side effects. In the present paper, we investigated the regulation of A 2A adenosine receptors induced by antipsychotic drugs, commonly acting as D 2 dopamine receptor antagonists, in a cellular model co-expressing both A 2A and D 2 receptors. Our data suggest that the treatment of cells with the classical antipsychotic haloperidol increased both the affinity and responsiveness of the A 2A receptor and also affected the degree of A 2A -D 2 receptor heterodimerisation. In contrast, an atypical antipsychotic, clozapine, had no effect on A 2A adenosine receptor parameters, suggesting that the two classes of drugs have different effects on adenosinedopamine receptor interaction. Modifications to A 2A adenosine receptors may play a significant role in determining cerebral adenosine effects during the chronic administration of antipsychotics in psychiatric diseases and may account for the efficacy of A 2A adenosine receptor ligands in pathologies associated with dopaminergic system dysfunction.