Neuronal Activity, TGFß-Signaling and Unpredictable Chronic Stress Modulate Transcription of Gadd45 Family Members and DNA Methylation in the Hippocampus.

Neuronal activity is altered in several neurological and psychiatric diseases. Upon depolarization not only neurotransmitters are released but also cytokines and other activators of signaling cascades. Unraveling their complex implication in transcriptional control in receiving cells will contribute to understand specific central nervous system (CNS) pathologies and will be of therapeutically interest. In this study we depolarized mature hippocampal neurons in vitro using KCl and revealed increased release not only of brain-derived neurotrophic factor (BDNF) but also of transforming growth factor beta (TGFB). Neuronal activity together with BDNF and TGFB controls transcription of DNA modifying enzymes specifically members of the DNA-damage-inducible (Gadd) family, Gadd45a, Gadd45b, and Gadd45g. MeDIP followed by massive parallel sequencing and transcriptome analyses revealed less DNA methylation upon KCl treatment. Psychiatric disorder-related genes, namely Tshz1, Foxn3, Jarid2, Per1, Map3k5, and Arc are transcriptionally activated and demethylated upon neuronal activation. To analyze whether misexpression of Gadd45 family members are associated with psychiatric diseases, we applied unpredictable chronic mild stress (UCMS) as established model for depression to mice. UCMS led to reduced expression of Gadd45 family members. Taken together, our data demonstrate that Gadd45 family members are new putative targets for UCMS treatments.

The FOXG1/FOXO/SMAD network balances proliferation and differentiation of cortical progenitors and activates Kcnh3 expression in mature neurons.

Transforming growth factor ß (TGFß)-mediated anti-proliferative and differentiating effects promote neuronal differentiation during embryonic central nervous system development. TGFß downstream signals, composed of activated SMAD2/3, SMAD4 and a FOXO family member, promote the expression of cyclin-dependent kinase inhibitor Cdkn1a. In early CNS development, IGF1/PI3K signaling and the transcription factor FOXG1 inhibit FOXO- and TGFß-mediated Cdkn1a transcription. FOXG1 prevents cell cycle exit by binding to the SMAD/FOXO-protein complex. In this study we provide further details on the FOXG1/FOXO/SMAD transcription factor network. We identified ligands of the TGFß- and IGF-family, Foxo1, Foxo3 and Kcnh3 as novel FOXG1-target genes during telencephalic development and showed that FOXG1 interferes with Foxo1 and Tgfß transcription. Our data specify that FOXO1 activates Cdkn1a transcription. This process is under control of the IGF1-pathway, as Cdkn1a transcription increases when IGF1-signaling is pharmacologically inhibited. However, overexpression of CDKN1A and knockdown of Foxo1 and Foxo3 is not sufficient for neuronal differentiation, which is probably instructed by TGFß-signaling. In mature neurons, FOXG1 activates transcription of the seizure-related Kcnh3, which might be a FOXG1-target gene involved in the FOXG1 syndrome pathology.

Neural deletion of Tgfbr2 impairs angiogenesis through an altered secretome.

Simultaneous generation of neural cells and that of the nutrient-supplying vasculature during brain development is called neurovascular coupling. We report on a transgenic mouse with impaired transforming growth factor ß (TGFß)-signalling in forebrain-derived neural cells using a Foxg1-cre knock-in to drive the conditional knock-out of the Tgfbr2. Although the expression of FOXG1 is assigned to neural progenitors and neurons of the telencephalon, Foxg1(cre/+);Tgfbr2(flox/flox) (Tgfbr2-cKO) mutants displayed intracerebral haemorrhage. Blood vessels exhibited an atypical, clustered appearance were less in number and displayed reduced branching. Vascular endothelial growth factor (VEGF) A, insulin-like growth factor (IGF) 1, IGF2, TGFß, inhibitor of DNA binding (ID) 1, thrombospondin (THBS) 2, and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 1 were altered in either expression levels or tissue distribution. Accordingly, human umbilical vein endothelial cells (HUVEC) displayed branching defects after stimulation with conditioned medium (CM) that was derived from primary neural cultures of the ventral and dorsal telencephalon of Tgfbr2-cKO. Supplementing CM of Tgfbr2-cKO with VEGFA rescued these defects, but application of TGFß aggravated them. HUVEC showed reduced migration towards CM of mutants compared with controls. Supplementing the CM with growth factors VEGFA, fibroblast growth factor (FGF) 2 and IGF1 partially restored HUVEC migration. In contrast, TGFß supplementation further impaired migration of HUVEC. We observed differences along the dorso-ventral axis of the telencephalon with regard to the impact of these factors on the phenotype. Together these data establish a TGFBR2-dependent molecular crosstalk between neural and endothelial cells during brain vessel development. These findings will be useful to further elucidate neurovascular interaction in general and to understand pathologies of the blood vessel system such as intracerebral haemorrhages, hereditary haemorrhagic telangiectasia, Alzheimers disease, cerebral amyloid angiopathy or tumour biology.

PI3K-p110-alpha-subtype signalling mediates survival, proliferation and neurogenesis of cortical progenitor cells via activation of mTORC2.

Development of the cerebral cortex is controlled by growth factors among which transforming growth factor beta (TGFß) and insulin-like growth factor 1 (IGF1) have a central role. The TGFß- and IGF1-pathways cross-talk and share signalling molecules, but in the central nervous system putative points of intersection remain unknown. We studied the biological effects and down-stream molecules of TGFß and IGF1 in cells derived from the mouse cerebral cortex at two developmental time points, E13.5 and E16.5. IGF1 induces PI3K, AKT and the mammalian target of rapamycin complexes (mTORC1/mTORC2) primarily in E13.5-derived cells, resulting in proliferation, survival and neuronal differentiation, but has small impact on E16.5-derived cells. TGFß has little effect at E13.5. It does not activate the PI3K- and mTOR-signalling network directly, but requires its activity to mediate neuronal differentiation specifically at E16.5. Our data indicate a central role of mTORC2 in survival, proliferation as well as neuronal differentiation of E16.5-derived cortical cells. mTORC2 promotes these cellular processes and is under control of PI3K-p110-alpha signalling. PI3K-p110-beta signalling activates mTORC2 in E16.5-derived cells but it does not influence cell survival, proliferation and differentiation. This finding indicates that different mTORC2 subtypes may be implicated in cortical development and that these subtypes are under control of different PI3K isoforms. Within developing cortical cells TGFß- and IGF-signalling activities are timely separated. TGFß dominates in E16.5-derived cells and drives neuronal differentiation. IGF influences survival, proliferation and neuronal differentiation in E13.5-derived cells. mTORC2-signalling in E16.5-derived cells influences survival, proliferation and differentiation, activated through PI3K-p110-alpha. PI3K-p110-beta-signalling activates a different mTORC2. Both PI3K/mTORC2-signalling pathways are required but not directly activated in TGFß-mediated neuronal differentiation.

Resilience to audiogenic seizures is associated with p-ERK1/2 dephosphorylation in the subiculum of Fmr1 knockout mice.

Young, but not adult, fragile X mental retardation gene (Fmr1) knockout (KO) mice display audiogenic seizures (AGS) that can be prevented by inhibiting extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation. In order to identify the cerebral regions involved in these phenomena, we characterized the response to AGS in Fmr1 KO mice and wild type (WT) controls at postnatal day (P) 45 and P90. To characterize the diverse response to AGS in various cerebral regions, we evaluated the activity markers FosB/ΔFosB and phosphorylated ERK1/2 (p-ERK1/2). Wild running (100% of tested mice) followed by clonic/tonic seizures (30%) were observed in P45 Fmr1 KO mice, but not in WT mice. In P90 Fmr1 KO mice, wild running was only present in 25% of tested animals. Basal FosB/ΔFosB immunoreactivity was higher (P < 0.01 vs. WT) in the CA1 and subiculum of P45 Fmr1 KO mice. Following the AGS test, FosB/ΔFosB expression consistently increased in most of the analyzed regions in both groups at P45, but not at P90. Interestingly, FosB/ΔFosB immunoreactivity was significantly higher in P45 Fmr1 KO mice in the medial geniculate body (P < 0.05 vs. WT) and CA3 (P < 0.01). Neurons presenting with immunopositivity to p-ERK1/2 were more abundant in the subiculum of Fmr1 KO mice in control condition (P < 0.05 vs. WT, in both age groups). In this region, p-ERK1/2-immunopositive cells significantly decreased (-75%, P < 0.01) in P90 Fmr1 KO mice exposed to the AGS test, but no changes were found in P45 mice or in other brain regions. In both age groups of WT mice, p-ERK1/2-immunopositive cells increased in the subiculum after exposure to the acoustic test. Our findings illustrate that FosB/ΔFosB markers are overexpressed in the medial geniculate body and CA3 in Fmr1 KO mice experiencing AGS, and that p-ERK1/2 is markedly decreased in the subiculum of Fmr1 KO mice resistant to AGS induction. These findings suggest that resilience to AGS is associated with dephosphorylation of p-ERK1/2 in the subiculum of mature Fmr1 KO mice.

Beneficial effects of desacyl-ghrelin, hexarelin and EP-80317 in models of status epilepticus.

It has been reported that ghrelin exerts anticonvulsive effects in models of epilepsy. In this study we aimed to characterize the anticonvulsive activity of ghrelin and other growth hormone secretagogue receptor 1a (GHSR(1a)) ligands in rats exposed to status epilepticus induced by pilocarpine or kainate. Firstly, in three independent experiments, before receiving pilocarpine (380 mg/kg, i.p.), rats were pretreated with one among ghrelin (1.5mg/kg), desacyl-ghrelin (1.5mg/kg), hexarelin (330 µg/kg), EP-80317 (330 µg/kg), JMV-1843 (330 µg/kg), JMV-2959 (330 µg/kg) or saline. Secondly, in the fourth experiment, rats were pretreated with i.p. ghrelin, desacyl-ghrelin, hexarelin, EP-80317 or saline, followed by kainate (15 mg/kg, i.p.). We evaluated: induction of generalized seizures, latency to generalized seizures, status epilepticus, latency to status epilepticus (the time lag between the first tonic-clonic convulsion and the switch to continuous seizures) and mortality. In the pilocarpine model, 60% of rats pretreated with EP-80317 (P<0.05) showed no seizure. Hexarelin and EP-80317 were both able to prevent progression to status epilepticus in pilocarpine-treated rats (P<0.05). When status epilepticus was induced by kainate, seizures developed with few exceptions. However, latency to status epilepticus was significantly (P<0.01) longer in rats pretreated with desacyl-ghrelin, whereas hexarelin and EP-80317 did not display any effect. Almost all GHSR(1a) ligands prevented pilocarpine-induced mortality, which was observed only in rats pretreated with saline or JMV-2959. After kainate administration, all rats survived to status epilepticus. These findings demonstrate that desacyl-ghrelin, hexarelin and EP-80317 but not other GHSR(1a) ligands display relevant anticonvulsive properties in models of limbic seizures.