Forkhead box g1 (Foxg1) is a nuclear-cytosolic transcription factor essential for the forebrain development and involved in neurodevelopmental and cancer pathologies. Despite the importance of this protein, little is known about the modalities by which it exerts such a large number of cellular functions. Here we show that a fraction of Foxg1 is localized within the mitochondria in cell lines, primary neuronal or glial cell cultures, and in the mouse cortex. Import of Foxg1 in isolated mitochondria appears to be membrane potential-dependent. Amino acids (aa) 277-302 were identified as critical for mitochondrial localization. Overexpression of full-length Foxg1 enhanced mitochondrial membrane potential (ΔΨm) and promoted mitochondrial fission and mitosis. Conversely, overexpression of the C-term Foxg1 (aa 272-481), which is selectively localized in the mitochondrial matrix, enhanced organelle fusion and promoted the early phase of neuronal differentiation. These findings suggest that the different subcellular localizations of Foxg1 control the machinery that brings about cell differentiation, replication, and bioenergetics, possibly linking mitochondrial functions to embryonic development and pathological conditions.Rett syndrome | autism | cancer | brain cortex | development

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...

Nerve growth factor (NGF) is a key mediator of nociception, acting during the development and differentiation of dorsal root ganglion (DRG) neurons, and on adult DRG neuron sensitization to painful stimuli. NGF also has central actions in the brain, where it regulates the phenotypic maintenance of cholinergic neurons. The physiological function of NGF as a pain mediator is altered in patients with Hereditary Sensory and Autonomic Neuropathy type V (HSAN V), caused by the 661CϾT transition in the Ngf gene, resulting in the R100W missense mutation in mature NGF. Homozygous HSAN V patients present with congenital pain insensitivity, but are cognitively normal. This led us to hypothesize that the R100W mutation may differentially affect the central and peripheral actions of NGF. To test this hypothesis and provide a mechanistic basis to the HSAN V phenotype, we generated transgenic mice harboring the human 661CϾT mutation in the Ngf gene and studied both males and females. We demonstrate that heterozygous NGF R100W/wt mice display impaired nociception. DRG neurons of NGF R100W/wt mice are morphologically normal, with no alteration in the different DRG subpopulations, whereas skin innervation is reduced. The NGF R100W protein has reduced capability to activate pain-specific signaling, paralleling its reduced ability to induce mechanical allodynia. Surprisingly, however, NGF R100W/wt mice, unlike heterozygous mNGF ϩ/Ϫ mice, show no learning or memory deficits, despite a reduction in secretion and brain levels of NGF. The results exclude haploinsufficiency of NGF as a mechanistic cause for heterozygous HSAN V mice and demonstrate a specific effect of the R100W mutation on nociception.

The Forkhead Box G1 (FOXG1) gene encodes a transcription factor with an essential role in mammalian telencephalon development. FOXG1-related disorders, caused by deletions, intragenic mutations or duplications, are usually associated with severe intellectual disability, autistic features, and, in 87% of subjects, epileptiform manifestations. In a subset of patients with FoxG1 mutations, seizures remain intractable, prompting the need for novel therapeutic options. To address this issue, we took advantage of a haploinsufficient animal model, the FoxG1 +/− mouse. In vivo electrophysiological analyses of FoxG1 +/− mice detected hippocampal hyperexcitability, which turned into overt seizures upon delivery of the proconvulsant kainic acid, as confirmed by behavioral observations. These alterations were associated with decreased expression of the chloride transporter KCC2. Next, we tested whether a triheptanoin-based anaplerotic diet could have an impact on the pathological phenotype of FoxG1 +/− mice. This manipulation abated altered neural activity and normalized enhanced susceptibility to proconvulsant-induced seizures, in addition to rescuing altered expression of KCC2 and increasing the levels of the GABA transporter vGAT. In conclusion, our data show that FoxG1 haploinsufficiency causes dysfunction of hippocampal circuits and increases the susceptibility to a proconvulsant insult, and that these alterations are rescued by triheptanoin dietary treatment.