Human induced pluripotent stem cells (hiPSCs) are emerging as a tool for understanding human brain development at cellular, molecular, and genomic levels. Here we show that hiPSCs grown in suspension in the presence of rostral neuralizing factors can generate 3D structures containing polarized radial glia, intermediate progenitors, and a spectrum of layer-specific cortical neurons reminiscent of their organization in vivo. The hiPSC-derived multilayered structures express a gene expression profile typical of the embryonic telencephalon but not that of other CNS regions. Their transcriptome is highly enriched in transcription factors controlling the specification, growth, and patterning of the dorsal telencephalon and displays highest correlation with that of the early human cerebral cortical wall at 8-10 wk after conception. Thus, hiPSC are capable of enacting a transcriptional program specifying human telencephalic (pallial) development. This model will allow the study of human brain development as well as disorders of the human cerebral cortex.human embryonic stem cell | embryo | differentiation | cortical layer E merging data highlight the complexity and dynamic nature of gene expression in the central nervous system (CNS) and the divergence between human and other mammalian species, which is especially pronounced in the developing brain (1-4). Exploring such differences may reveal the genetic underpinnings of the larger size and complex architecture of the human brain and elucidate the molecular and cellular substrates of higher cognitive functions, as well as of our vulnerability to neurodevelopmental and neurodegenerative disorders. To understand the genetic programs that drive cell specification and differentiation in the human brain, it is important to develop model systems that recapitulate dynamic aspects of neural development, in addition to making inferences from commonly used models of lower mammalian species.Recapitulating human neural development in vitro using human induced pluripotent stem cells (hiPSCs) can provide our first understanding of how genetic variation and disease-causing mutations influence neural development. Human iPSCs generated from reprogrammed cells can be differentiated into any tissue, including the CNS, while maintaining the genetic background of the individual of origin. These critical features have been exploited to model monogenic forms of neurodevelopmental disorders, such as Rett and Timothy syndromes, and even psychiatric disorders with complex inheritance, such as schizophrenia (5-7). The brain and spinal cord develop according to distinct differentiation programs from the earliest stages of CNS development (i.e., at the progenitor stage during gastrulation) (8, 9). Regional differences in gene expression within stem and progenitor cells appear at the onset of the formation of both mouse (10, 11) and human CNS, as shown by recent studies of the human transcriptome using postmortem tissue (4).Neural cells are thought to differentiate by "default" into an anterior, for...

The association of the histone deacetylase (HDAC) inhibitor valproate (VPA) with atypical antipsychotics has become a frequent treatment strategy for schizophrenia and bipolar disorder. Because the VPA doses administered are elevated, one cannot assume that the benefits of the VPA plus antipsychotic treatment are exclusively related to the covalent modifications of nucleosomal histone tails. We compared the actions of N-(2-aminophenyl)-4-[N-(pyridin-3-yl-methoxycarbonyl)aminomethyl]benzamide derivative (MS-275), which is a potent HDAC inhibitor in vitro, with the actions of VPA for their ability to (i) increase the acetylated status of brain nucleosomal histone tail domains and (ii) to regulate brain histone-RELN and histone-GAD67 promoter interactions. MS-275 increases the content of acetylhistone 3 (Ac-H3) in the frontal cortex. Whereas this response peaks after a s.c. injection of 15 mol͞kg, the increase in Ac-H3 content in the hippocampus becomes significant only after an injection of 60 mol͞kg, suggesting that MS-275 is 30-to 100-fold more potent than VPA in increasing Ac-H3 in these brain regions. In contrast to VPA, MS-275, in doses up to 120 mol͞kg, fails to increase Ac-H3 content in the striatum. Chromatin immunoprecipitation shows that MS-275 increases Ac-H3-RELN and Ac-H3-GAD67 promoter interaction in the frontal cortex. These results suggest that MS-275 is a potent brain region-selective HDAC inhibitor. It is likely that, in addition to MS-275, other benzamide derivatives, such as sulpiride, are brain-region selective inhibitors of HDACs. Hence, some benzamide derivatives may express a greater efficacy than VPA as an adjunctive to antipsychotics in the treatment of epigentically induced psychiatric disorders. bipolar disorder ͉ reelin ͉ schizophrenia ͉ histone code ͉ chromatin remodeling A prefrontal cortex GABAergic neuron dysfunction, which is characterized by a reduction of the 67-kDa form of glutamic acid decarboxylase (GAD 67 ) and reelin expression, is one of the most consistent neuropathological findings in postmortem brain studies of schizophrenia (SZ) and bipolar (BP) disorder (1-9). These expression deficits cannot be explained by reelin or GAD 67 gene haploinsufficiency (10, 11). Converging epidemiological (12), histological, and biochemical (10, 13-17) evidence suggests that the pathogenesis of this dysfunction may be related to a disruption of epigenetic signaling, resulting in the selective hypermethylation of several GABAergic gene promoters that characterize SZ as a selective defect of gene transcription in GABAergic cortical neurons (13,14). Such hypermethylation is very likely mediated by the overexpression of DNA methyltransferase 1 (DNMT1) (15, 18), which has been found to be operative in cortical and subcortical GABAergic interneurons of SZ and BP patients.The long-term objective of this line of research is to identify drugs that selectively correct a basic defect of SZ, which is an epigenetic GABAergic neuron dysfunction, by directly or indirectly reducing the RELN and GAD 67 pr...

Reduction of prefrontal cortex glutamic acid decarboxylase (GAD 67) and reelin (mRNAs and proteins) expression is the most consistent finding reported by several studies of postmortem schizophrenia (SZ) brains. Converging evidence suggests that the reduced GAD 67 and reelin expression in cortical GABAergic interneurons of SZ brains is the consequence of an epigenetic hypermethylation of RELN and GAD 67 promoters very likely mediated by the overexpression of DNA methyltransferase 1 in cortical GABAergic interneurons. Studies of the molecular mechanisms (DNA methylation plus related chromatin remodeling factors) that cause the down-regulation of reelin and GAD 67 in SZ brains have important implications not only to understand the disease pathogenesis but also to improve present pharmacological interventions to treat SZ. The mouse treated with L-methionine models some of the molecular neuropathologies detected in SZ, including the hypermethylation of RELN promoter CpG islands and the down-regulation of reelin and GAD 67 expression. We now report that in these mice, RELN and GAD 67 promoters express an increased recruitment of methyl-CpG binding domain proteins. In these mice the histone deacetylase inhibitor valproate, which increases acetylated histone content in cortical GABAergic interneurons, also prevents METinduced RELN promoter hypermethylation and reduces the methylCpG binding domain protein binding to RELN and GAD 67 promoters. These findings suggest that DNA hypermethylation and the associated chromatin remodeling may be critically important in mediating the epigenetic down-regulation of reelin and GAD 67 expression detected in cortical GABAergic interneurons of SZ patients.DNA methyltransferase1 ͉ L-methionine ͉ methyl binding domain proteins ͉ valproate ͉ chromatin S chizophrenia (SZ) pathophysiology is characterized by a down-regulation of several GABAergic neuronal markers including GAD 67 and reelin mRNAs and proteins (1-8).Reelin is an extracellular matrix protein, synthesized and secreted from cortical GABAergic interneurons (9-12), that surrounds apical and basal dendritic spines of pyramidal cortical neurons (13-14). This protein not only plays a defined role in prenatal central nervous system development (13-15) but also plays an important role in the adult brain by modulating cortical pyramidal neuron dendritic spine expression density, the branching of dendrites, and the expression of long-term potentiation (14,16,17). Very likely, reelin has a role in regulating the event-related increase of protein synthesis mediated by the dendritic translation of cytosolic mRNAs (18). In this scenario, the down-regulation of reelin expression in neocortices and hippocampi of SZ patients (SZP) (1, 5, 19) may be important in mediating the down-regulation of pyramidal neuron dendritic branching and spine expression and in the neuropil hypoplasticity typical of SZ (20)(21)(22).GAD 67 is one of two molecular forms of the GABA synthesizing enzymes expressed in GABAergic interneurons (23). In SZP, the down-regula...

Glial fibrillary acidic protein (GFAP)+ cells give rise to new neurons in the neurogenic niches; whether they are able to generate neurons in the cortical parenchyma is not known. Here, we use genetic fate mapping to examine the progeny of GFAP+ cells after postnatal hypoxia, a model for the brain injury observed in premature children. Following hypoxia, immature cortical astroglia underwent a shift towards neuronal fate and generated cortical excitatory neurons that appeared synaptically integrated into the circuitry. Fate mapped cortical GFAP+ cells derived ex vivo from hypoxic, but not normoxic mice were able to form pluripotent, long-term self-renewing neurospheres. Similarly, exposure to low oxygen conditions in vitro induced stem cell-like potential in immature cortical GFAP+ cells. Our data support the conclusion that hypoxia promotes pluripotency in GFAP+ cells in the cortical parenchyma. Such plasticity possibly explains the cognitive recovery found in some preterm children.

The endogenous neurotransmitter noradrenaline (NA) is known to exert potent anti-inflammatory effects in glial cells, as well as provide neuroprotection against excitatory and inflammatory stimuli. These properties raise the possibility that increasing levels of NA in the central nervous system (CNS) could provide benefit in neurological diseases and conditions containing an inflammatory component. In the current study, we tested this possibility by examining the consequences of selectively modulating CNS NA levels on the development of clinical signs in experimental autoimmune encephalomyelitis (EAE). In mice immunized with myelin oligodendrocyte glycoprotein peptide to develop a chronic disease, pretreatment to selectively deplete CNS NA levels exacerbated clinical scores. Elevation of NA levels using the selective NA reuptake inhibitor atomoxetine did not affect clinical scores, while treatment of immunized mice with the synthetic NA precursor L-threo-3,4-dihydroxyphenylserine (L-DOPS) prevented further worsening. In contrast, treatment of mice with a combination of atomoxetine and L-DOPS led to significant improvement in clinical scores as compared to the control group. The combined treatment reduced astrocyte activation in the molecular layer of the cerebellum as assessed by staining for glial fibrillary protein but did not affect Th1 or Th17 type cytokine production from splenic T cells. These data suggest that selective elevation of CNS NA levels could provide benefit in EAE and multiple sclerosis without influencing peripheral immune responses.

In EAE (experimental autoimmune encephalomyelitis), agonists of PPARs (peroxisome proliferator-activated receptors) provide clinical benefit and reduce damage. In contrast with PPARγ, agonists of PPARδ are more effective when given at later stages of EAE and increase myelin gene expression, suggesting effects on OL (oligodendrocyte) maturation. In the present study we examined effects of the PPARδ agonist GW0742 on OPCs (OL progenitor cells), and tested whether the effects involve modulation of BMPs (bone morphogenetic proteins). We show that effects of GW0742 are mediated through PPARδ since no amelioration of EAE clinical scores was observed in PPARδ-null mice. In OPCs derived from E13 mice (where E is embryonic day), GW0742, but not the PPARγ agonist pioglitazone, increased the number of myelin-producing OLs. This was due to activation of PPARδ since process formation was reduced in PPARδ-null compared with wild-type OPCs. In both OPCs and enriched astrocyte cultures, GW0742 increased noggin protein expression; however, noggin mRNA was only increased in astrocytes. In contrast, GW0742 reduced BMP2 and BMP4 mRNA levels in OPCs, with lesser effects in astrocytes. These findings demonstrate that PPARδ plays a role in OPC maturation, mediated, in part, by regulation of BMP and BMP antagonists.

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Glioma immunosuppression includes the secretion of cytokines that down-regulate the host immune response resulting in tumor survival. The mechanisms of cytokine-induced immunosuppression are not well understood and are considered in this study. Glioma cells were incubated with supernatant from activated and naïve T-cells. A separate culture of T-cells (naïve, CD3-activated, and CD3/CD28 activated) was then incubated with conditioned media from the treated glioma cells as well as individual and combination recombinant cytokines. These T-cells were tested for viability, proliferation and IFN-􀀁 release. Several conclusions were drawn from these experiments: cytotoxicity is not a means of glioma immunosuppression, glioma conditioned media decreases proliferation of CD3/CD28 activated T-cells acting potentially through IL10 and IGFBP, and these cytokines also decrease IFN-􀀁 secretion from all varieties of T-cells suggesting that T-cell differentiation away from TH1 is another potential means of immunosuppression. These results necessitate further analysis of proliferation and differentiation as potential mechanisms of immunosuppression and the incorporation of this knowledge into the production of a more efficacious tumor vaccine.