A triheptanoin-supplemented diet rescues hippocampal hyperexcitability and seizure susceptibility in FoxG1+/- mice

The Forkhead Box G1 (FOXG1) gene encodes a transcription factor with an essential role in the 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 at least a subset of the 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 the enhanced susceptibility to proconvulsant-induced seizures, in addition to rescuing the 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

Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics.

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

IκB-α Represses the Transcriptional Activity of the HIV-1 Tat Transactivator by Promoting Its Nuclear Export

The long terminal repeat of human immunodeficiency virus, type 1 (HIV-1) contains an NF-kappaB enhancer and is potently inhibited by IkappaB-alphaS32/36A, a proteolysis-resistant inhibitor of NF-kappaB transacting factors. The evidence that NF-kappaB is dispensable for HIV-1 expression raises the question of whether IkappaB-alpha represses the HIV-1 transcription by mechanisms distinct from NF-kappaB inhibition. Here, we report that IkappaB-alpha negatively regulates the HIV-1 expression and replication in an NF-kappaB-independent manner by directly binding to Tat, which results in the nuclear export and cytoplasmic sequestration of the viral transactivator. The sequence of IkappaB-alpha required for Tat inhibition spans from amino acids 72 to 287 and includes the nuclear localization signal, the carboxyl-terminal nuclear export signal, and the binding site for the arginine-rich domain of Tat. This novel mechanism of cross-talk between Tat and IkappaB-alpha provides further insights into the mechanisms of HIV-1 regulation and could assist in the development of novel strategies for AIDS therapy

Cortical Seizures in FoxG1+/− Mice are Accompanied by Akt/S6 Overactivation, Excitation/Inhibition Imbalance and Impaired Synaptic Transmission

The correct morphofunctional shaping of the cerebral cortex requires a continuous interaction between intrinsic (genes/molecules expressed within the tissue) and extrinsic (e.g., neural activity) factors at all developmental stages. Forkhead Box G1 (FOXG1) is an evolutionarily conserved transcription factor, essential for the cerebral cortex patterning and layering. FOXG1-related disorders, including the congenital form of Rett syndrome, can be caused by deletions, intragenic mutations or duplications. These genetic alterations are associated with a complex phenotypic spectrum, spanning from intellectual disability, microcephaly, to autistic features, and epilepsy. We investigated the functional correlates of dysregulated gene expression by performing electrophysiological assays on FoxG1+/− mice. Local Field Potential (LFP) recordings on freely moving animals detected cortical hyperexcitability. On the other hand, patch-clamp recordings showed a downregulation of spontaneous glutamatergic transmission. These findings were accompanied by overactivation of Akt/S6 signaling. Furthermore, the expression of vesicular glutamate transporter 2 (vGluT2) was increased, whereas the level of the potassium/chloride cotransporter KCC2 was reduced, thus indicating a higher excitation/inhibition ratio. Our findings provide evidence that altered expression of a key gene for cortical development can result in specific alterations in neural circuit function at the macro- and micro-scale, along with dysregulated intracellular signaling and expression of proteins controlling circuit excitability

Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics.

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

Imbalance of excitatory/inhibitory synaptic protein expression in iPSC-derived neurons from FOXG1^+/- patients and in foxg1^+/- mice

Rett syndrome (RTT) is a severe neurodevelopmental disorder associated with mutations in either MECP2, CDKL5 or FOXG1. The precise molecular mechanisms that lead to the pathogenesis of RTT have yet to be elucidated. We recently reported that expression of GluD1 (orphan glutamate receptor δ-1 subunit) is increased in iPSC-derived neurons obtained from patients with mutations in either MECP2 or CDKL5. GluD1 controls synaptic differentiation and shifts the balance between excitatory and inhibitory synapses toward the latter. Thus, an increase in GluD1 might be a critical factor in the etiology of RTT by affecting the excitatory/inhibitory balance in the developing brain. To test this hypothesis, we generated iPSC-derived neurons from FOXG1(+/−) patients. We analyzed mRNA and protein levels of GluD1 together with key markers of excitatory and inhibitory synapses in these iPSC-derived neurons and in Foxg1(+/−) mouse fetal (E11.5) and adult (P70) brains. We found strong correlation between iPSC-derived neurons and fetal mouse brains, where GluD1 and inhibitory synaptic markers (GAD67 and GABA AR-α1) were increased, whereas the levels of a number of excitatory synaptic markers (VGLUT1, GluA1, GluN1 and PSD-95) were decreased. In adult mice, GluD1 was decreased along with all GABAergic and glutamatergic markers. Our findings further the understanding of the etiology of RTT by introducing a new pathological event occurring in the brain of FOXG1(+/−) patients during embryonic development and its time-dependent shift toward a general decrease in brain synapses

The use of multilayer nano-encapsulation for the immunoprotection of isolated human islets: in vitro and in vivo studies.

In an attempt to improve the outcome of pancreatic islet transplantation, we performed in-vitro and in-vivo experiments with isolated human islets coated by multi-layer nano-encapsulation. Human islets were isolated from 32 non-diabetic donors (age: 63±17 yrs, BMI: 26.8 ± 3.5 kg/m2, M/F: 17/15) by enzymatic digestion and gradient purifi cation. Multi-layer nano-encapsulation was performed by electrostatic binding of differently charged polymers [chitosan and poly(sodium styrene sulfonate)], up to 9 layers. Morphological, ultrastructural, functional and transplantation studies where then accomplished with the nano-coated human islets. The procedure provided full coating of the human islets (thickness: 104.2±4.2 nm), as assessed by fl uorescence, confocal and electron microscopy (EM). Vital staining and EM showed ≥90% cell survival and well maintained beta and alpha cell ultrastructure, with unchanged morphology and morphometry of intracellular organelles. Insulin secretion from nanoencapsulated islets was 44±33 μU/ml (mean±SD) at 3.3 mM glucose and increased to 176±120 μU/ml at 16.7 mM glucose (p<0.01), with a stimulation index of 4.6±2.7. Perifusion studies showed maintained dynamics of insulin secretion. Toxicity by palmitate or cytokine exposure was signifi cantly reduced in nano-coated islets. Transplantation of nano-encapsulated islets under the kidney capsule of C57Bl/6J mice with streptozotocin-induced diabetes allowed long term normal or near normal glycemia, with intra-peritoneal glucose tolerance test results similar to those of non-diabetic mice. Light and electron microscopy of nano-coated islet grafts was performed at one month post-transplantation, showing minimal mononuclear cell infi ltration, preservation of islet cell ultrastructure and signs of revascularization. Isolated human islets were effi ciently encapsulated by this multi-layer nano-coating approach, with preserved in-vitro and in-vivo function. Supported By: CariPisa Foundatio