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Astrocytes are the most abundant cell type in the human brain and are important regulators of several critical cellular functions, including synaptic transmission. Although astrocytes are known to play a central role in the etiology and pathophysiology of schizophrenia, little is known about their potential involvement in clinical response to the antipsychotic clozapine. Moreover, astrocytes display a remarkable degree of morphological diversity, but the potential contribution of astrocytic subtypes to disease biology and drug response has received little attention. Here, we used state-of-the-art human induced pluripotent stem cell (hiPSC) technology to derive a morphological subtype of astrocytes from healthy individuals and individuals with schizophrenia, including responders and nonresponders to clozapine. Using functional assays and transcriptional profiling, we identified a distinct gene expression signature highly specific to schizophrenia as shown by disease association analysis of more than 10 000 diseases. We further found reduced levels of both glutamate and the NMDA receptor coagonist d-serine in subtype astrocytes derived from schizophrenia patients, and that exposure to clozapine only rescued this deficiency in cells from clozapine responders, providing further evidence that d-serine in particular, and NMDA receptor-mediated glutamatergic neurotransmission in general, could play an important role in disease pathophysiology and clozapine action. Our study represents a first attempt to explore the potential contribution of astrocyte diversity to schizophrenia pathophysiology using a human cellular model. Our findings suggest that specialized subtypes of astrocytes could be important modulators of disease pathophysiology and clinical drug response, and warrant further investigations.

The biological functions of primate-specific astroglia, interlaminar astrocytes (IAs), remain enigmatic due to the lack of access to experimental materials, especially in humans. Here, we develop a protocol for the derivation of IAs from healthy normal and schizophrenia patient (SCZ)-specific human pluripotent stem cells. We find that IAs possess a functional profile which is distinct from that of protoplasmic astrocytes, particularly in synapse-associated functions such as neuroactive ligand-receptor interactions, calcium signaling, and long-term potentiation. These synaptic functions are further corroborated by our findings that IAs respond to glutamate, which mediates Ca2+ signaling and in turn leads to the release of gliotransmitters to interact with neurons and astrocytes. We further show that SCZ IAs shift their functional characteristic from synaptic signaling to Matrix-associated functions, with a notable impairment in the levels of gliotransmitters. We anticipate that these findings will prompt more penetrating investigations into the role of IAs in glia biology and brain disorders.

Neural rosette formation is a critical morphogenetic process during neural development, whereby neural stem cells are enclosed in rosette niches to equipoise proliferation and differentiation. How neural rosettes form and provide a regulatory micro-environment remains to be elucidated. We employed the human embryonic stem cell-based neural rosette system to investigate the structural development and function of neural rosettes. Our study shows that neural rosette formation consists of 5 types of cell movements: intercalation, constriction, polarization, elongation, and lumen formation. Ca2+ signaling plays a pivotal role in the five steps by regulating the actions of the cytoskeletal complexes, ACTIN, MYOSIN II, and TUBULIN during intercalation, constriction, and elongation. These in turn control the polarizing elements, ZO-1, PARD3, and β-CATENIN during polarization and lumen formation in neural rosette formation. We further demonstrated that the dismantlement of neural rosettes, mediated by the destruction of cytoskeletal elements, promoted neurogenesis and astrogenesis prematurely, indicating that an intact rosette structure is essential for orderly neural development.

Neural rosette formation is a critical morphogenetic process during neural development, whereby neural stem cells are enclosed in rosette niches to equipoise proliferation and differentiation. How neural rosettes form and provide a regulatory micro-environment remains to be elucidated. We employed the human embryonic stem cell-based neural rosette system to investigate the structural development and function of neural rosettes. Our study shows that neural rosette formation consists of five types of morphological change: intercalation, constriction, polarization, elongation and lumen formation. Ca

Neural induction is the first step in the formation of the vertebrate central nervous system. The emerging consensus of the mechanisms underling neural induction is the combined influences from inhibiting bone morphogenetic protein (BMP) signaling and activating fibroblast growth factor (FGF)/Erk signaling, which act extrinsically via either autocrine or paracrine fashions. However, do intrinsic forces (cues) exist and do they play decisive roles in neural induction? These questions remain to be answered. Here, we have identified a novel neural initiator, neuronatin (Nnat), which acts as an intrinsic factor to promote neural fate in mammals and Xenopus. ESCs lacking this intrinsic factor fail to undergo neural induction despite the inhibition of the BMP pathway. We show that Nnat initiates neural induction in ESCs through increasing intracellular Ca2+ ([Ca2+]i) by antagonizing Ca2+-ATPase isoform 2 (sarco/endoplasmic reticulum Ca2+-ATPase isoform 2) in the endoplasmic reticulum, which in turn increases the phosphorylation of Erk1/2 and inhibits the BMP4 pathway and leads to neural induction in conjunction with FGF/Erk pathway.

Major programs in psychiatric genetics have identified 4150 risk loci for psychiatric disorders. These loci converge on a small\ud number of functional pathways, which span conventional diagnostic criteria, suggesting a partly common biology underlying\ud schizophrenia, autism and other psychiatric disorders. Nevertheless, the cellular phenotypes that capture the fundamental features\ud of psychiatric disorders have not yet been determined. Recent advances in genetics and stem cell biology offer new prospects for\ud cell-based modeling of psychiatric disorders. The advent of cell reprogramming and induced pluripotent stem cells (iPSC) provides\ud an opportunity to translate genetic findings into patient-specific in vitro models. iPSC technology is less than a decade old but holds\ud great promise for bridging the gaps between patients, genetics and biology. Despite many obvious advantages, iPSC studies still\ud present multiple challenges. In this expert review, we critically review the challenges for modeling of psychiatric disorders, potential\ud solutions and how iPSC technology can be used to develop an analytical framework for the evaluation and therapeutic\ud manipulation of fundamental disease processes.

Differentiation of pluripotent embryonic stem (ES) cells through multipotent neural stem (NS) cells into differentiated neurons is accompanied by wholesale changes in transcriptional programs. One factor that is present at all three stages and a key to neuronal differentiation is the RE1-silencing transcription factor (REST/NRSF). Here, we have used a novel chromatin immunoprecipitation-based cloning strategy (SACHI) to identify 89 REST target genes in ES cells, embryonic hippocampal NS cells and mature hippocampus. The gene products are involved in all aspects of neuronal function, especially neuronal differentiation, axonal growth, vesicular transport and release, and ionic conductance. Most target genes are silent or expressed at low levels in ES and NS cells, but are expressed at much higher levels in hippocampus. These data indicate that the REST regulon is specific to each developmental stage and support the notion that REST plays distinct roles in regulating gene expression in pluripotent ES cells, multipotent NS cells, and mature neurons.

We developed a model system whereby HP1 can be targeted to pericentric heterochromatin in ES cells lacking Suv(3)9h1/2 histone methyltransferase (HMTase) activities. HP1 so targeted can reconstitute tri-methylated lysine 9 of histone H3 (Me(3)K9H3) and tri-methylated lysine 20 of histone H4 (Me(3)K20H4) at pericentric heterochromatin, indicating that HP1 can regulate the distribution of these histone modifications in vivo. Both homo- and hetero-typic interactions between the HP1 isotypes were demonstrated in vivo as were HP1 interactions with the ESET/SETDB1 HMTase and the ATRX chromatin remodelling enzyme. We conclude that HP1 not only "deciphers" the histone code but can also "encode it".

In late sepsis, it has been established that the liver plays a major role in the initiation of multiorgan failure, which is the most lethal complication in hospitals. The molecular mechanism underlying liver failure that results from sepsis remains elusive. This study was undertaken to identify the bona fide differentially expressed genes in the 18-h septic liver by suppression subtractive hybridization, and the data were corroborated by Northern blot analysis. The differential gene expression profile renders a clue as to the genes involved in septic liver failure. The cecal ligation and puncture (CLP) model of a polymicrobial septic rat was used, with the late sepsis referring to animals sacrificed at 18 h after CLP. We have identified three upregulated genes (TII-kininogen, serine protease inhibitor 2.2 [Spi2.2], and alpha 2 macroglobulin [alpha M]) and six down-regulated genes (hydroxysteroid dehydrogenase [3 alpha HSD], EST189895/mouse RNase4, bile acid-CoA-amino acid N-acyltransferase [kan-1/rBAT], IF1, albumin, and alpha 2u-globulins [alpha 2u-G PGCL1]). Among these genes, the 3 alpha HSD and kan-1/rBAT are involved in bile acid metabolism. The IF1 plays a crucial role in any disease that involves ATP hydrolysis by F1F0-ATPase. The alpha 2M, TII-kininogen, and Spi2.2 are protease inhibitors. The functions of the alpha 2u-G PGCL1 and EST189895/mouse RNase4 genes are unknown. The present results suggest that the roles of disturbance of bile acid metabolism/synthesis and the abolishment of ATP production may contribute to liver failure during late sepsis.

Mammalian receptors for gonadotropin-releasing hormone (GnRH) have over 85% sequence homology and similar ligand selectivity. Biological studies indicated that the chicken GnRH receptor has a distinct pharmacology, and certain antagonists of mammalian GnRH receptors function as agonists. To explore the structural determinants of this, we have cloned a chicken pituitary GnRH receptor and demonstrated that it has marked differences in primary amino acid sequence (59% homology) and in its interactions with GnRH analogs. The chicken GnRH receptor had high affinity for mammalian GnRH (K(i) 4.1 +/- 1.2 nM), similar to the human receptor (K(i) 4.8 +/- 1.2 nM). But, in contrast to the human receptor, it also had high affinity for chicken GnRH ([Gln(8)]GnRH) and GnRH II ([His(5),Trp(7),Tyr(8)]GnRH) (K(i) 5.3 +/- 0.5 and 0.6 +/- 0.01 nM). Three mammalian receptor antagonists were also pure antagonists in the chicken GnRH receptor. Another three, characterized by D-Lys(6) or D-isopropyl-Lys(6) moieties, functioned as pure antagonists in the human receptor but were full or partial agonists in the chicken receptor. This suggests that the Lys side chain interacts with functional groups of the chicken GnRH receptor to stabilize it in the active conformation and that these groups are not available in the activated human GnRH receptor. Substitution of the human receptor extracellular loop two with the chicken extracellular loop two identified this domain as capable of conferring agonist activity to mammalian antagonists. Although functioning of antagonists as agonists has been shown to be species-dependent for several GPCRs, the dependence of this on an extracellular domain has not been described.

The porcine perinatal myosin heavy chain (MyHC) is a major isoform in foetal skeletal muscles. We report here on its cDNA and genomic isolation, molecular characterisation and expression. Exon 2 and the first 4 bases of exon 3 of the perinatal MyHC gene, both part of the 5′-end untranslated region, showed differential splicing. About 2% of all perinatal MyHC transcripts of a 50-day-old foetus were without exon 2 and about half were without the 4 bases at the 5′-end of exon 3. Perinatal MyHC mRNA was expressed in all hind limb muscles of a 45-day-old foetus along with the slow and embryonic MyHC isoforms in the same fibres. Unlike other sarcomeric MyHCs reported to date, the porcine perinatal promoter is clustered with repeat elements (4 SINEs and 1 microsatellite) and is without a consensus TATA box at the predicted site upstream of exon 1. Nonetheless, in reporter gene transfections, its promoter was found to be highly muscle-specific. The absence of a TATA box may point to a fundamental difference in the regulatory function between the perinatal and adult MyHC isoforms.

We report on the cloning of the full-length complementary DNA for the chicken TRH receptor. Although the TRH receptor has been cloned from several mammalian species, this is the first report from another vertebrate class. The ligand binding pocket, which is situated in the transmembrane helices of the mouse and rat TRH receptors, is completely conserved in the chicken receptor. Pharmacological studies (receptor binding and signaling) employing several TRH analogs revealed that there are no significant differences between the chicken and mouse receptors. These findings show that there have been considerable evolutionary constraints on TRH receptor structure and function. Several truncated forms of the chicken TRH receptor that appear to retain a part of an intron and are truncated in the putative third intracellular loop were also cloned, but were nonfunctional. This study provides a useful tool for further studies on the roles of TRH in avian growth and TSH regulation.

SummaryDuring embryogenesis, the Activin/Nodal pathway promotes the mesendodermal lineage and inhibits neural fate. The molecular mechanisms underlying this role of the Activin/Nodal pathway are not clear. In this study, we report a role for protein tyrosine phosphatase 1B (PTP1B) in Activin-mediated early fate decisions during ESC differentiation and show that PTP1B acts as an effector of the Activin pathway to specify mesendodermal or neural fate. We found that the Activin/ALK4 pathway directly recruits PTP1B and stimulates its release from the endoplasmic reticulum through ALK4-mediated cleavage. Subsequently, PTP1B suppresses p-ERK1/2 signaling to inhibit neural specification and promote mesendodermal commitment. These findings suggest that a noncanonical Activin signaling pathway functions in lineage specification of mouse and human embryonic stem cells.

Neural development from blastocysts is strictly controlled by intricate transcriptional programmes that initiate the down-regulation of pluripotent genes, Oct4, Nanog and Rex1 in blastocysts followed by up-regulation of lineage-specific genes as neural development proceeds. Here, we demonstrate that the expression pattern of the transcription factor Rest mirrors those of pluripotent genes during neural development from embryonic stem (ES) cells and an early abrogation of Rest in ES cells using a combination of gene targeting and RNAi approaches causes defects in this process. Specifically, Rest ablation does not alter ES cell pluripotency, but impedes the production of Nestin(+) neural stem cells, neural progenitor cells and neurons, and results in defective adhesion, decrease in cell proliferation, increase in cell death and neuronal phenotypic defects typified by a reduction in migration and neurite elaboration. We also show that these Rest-null phenotypes are due to the dysregulation of its direct or indirect target genes, Lama1, Lamb1, Lamc1 and Lama2 and that these aberrant phenotypes can be rescued by laminins.

The chicken GnRH receptor (cGnRH-R) differs from all mammalian GnRH-Rs in possessing a cytoplasmic carboxyl-terminal tail. We have previously demonstrated that the cGnRH-R undergoes more rapid agonist-induced internalization than the mammalian GnRH-Rs and requires the carboxyl-terminal tail for this process. To investigate the structural determinants mediating this rapid internalization, a series of mutant receptors was generated, including progressive truncations of the tail and substitution of serine and threonine residues with alanine. Truncation of the carboxyl-terminal tail to position 366 and then to position 356 resulted in a progressive attenuation of the rate and total extent of receptor internalization. However, truncation between positions 356 and 346 did not alter the kinetics of internalization further, whereas a further truncation to position 337 resulted in an additional marked reduction of internalization. We show that the membrane-proximal Cys328 and the Thr369Thr370 doublet located in the distal carboxyl terminus play a critical role in mediating rapid internalization. We demonstrate that the cGnRH-R, when expressed in both COS-7 and HEK 293 cells, preferentially undergoes rapid agonist-induced internalization in a caveolae-like, dynamin-dependent manner. These conclusions are based on our observation that pretreatments with filipin and methyl-β-cyclodextrin, agents that disrupt lipid rafts such as caveolae, and coexpression of dominant-negative dynamin-1 (K44A) and caveolin-1 (Δ1–81) mutants, effectively inhibited rapid agonist-induced internalization. Furthermore, cGnRH-Rs appeared to be mobilized to the β-arrestin- and clathrin-coated, vesicle-mediated endocytic pathway upon β-arrestin overexpression.

Establishment and maintenance of the pluripotent state of ESCs is a key issue in stem cell biology and regenerative medicine, and consequently identification of transcription factors that regulate ESC pluripotency is an important goal. Singh et al. claim that the transcriptional repressor REST is such a regulator and that a 50% reduction of REST in ESCs leads to activation of a specific microRNA, miR-21, and that this subsequently results in loss of pluripotency markers and a reciprocal gain in some lineage-specific differentiation markers. In contrast, we show that, in haplodeficient Rest(+/-) ESCs, we detected no change in pluripotency markers, no precocious expression of differentiated neuronal markers and no interaction of REST with miR-21. It is vital that identification of factors that regulate pluripotency is based on robust, consistent data, and the contrast in data reported here undermines the claim by Singh et al. that REST is such a regulator.

The majority of vertebrates have two or three forms of gonadotropin-releasing hormone (GnRH), which appear to have arisen by successive gene duplication events. This suggests the possibility of concordant gene duplications of the GnRH receptor to produce two or more cognate receptors. Since the extracellular loop 3 (EC3) domain of mammalian GnRH receptors plays a role in distinguishing the different forms of GnRH, we have contemplated that the sequence of this domain will differ significantly in the putative cognate receptors. Degenerate oliognucleotides encoding the sequences of the transmembrane domains preceding and following EC3 were used for PCR amplification of genomic DNA from zebrafish ( Brachydanio rerio ), goldfish ( Carassius auratus ), African clawed frog ( Xenopus laevis ), chicken ( Gallus domesticus ), and lizard ( Agama atra ). Isolation and sequencing of specific clones revealed that they fell into three groups. Two of these were most similar to the mammalian pituitary GnRH receptor and were therefore designated Type IA and Type IB. The third form (designated Type II) was most different from the others and was identified in Xenopus, lizard, and human DNA. These findings support the concept of the existence of three distinct GnRH receptors, which have evolved in conjunction with three distinct GnRH ligand classes present in many vertebrates.

L-monosodium glutamate (MSG) (4 mg/g b.wt.) was injected subcutaneously to pups on days 1 and 3. At age 3.5 months, sexual and ingestive behaviors were observed. Neonatal MSG treatment resulted in severe and widespread neuron destruction in the basomedial hypothalamus of both sexes, but only superchiasmatic nucleus in male rats. A decline in sexual behavior was also observed. Serum levels of testosterone and dihydrotestosterone in male rats as well as serum level of progesterone in female rats were decreased. Ovariectomized MSG-treated female rats injected with estradiol benzoate followed by progesterone showed a dramatic improvement in sexual behavior, whereas castrated MSG-treated male rats injected with testosterone propionate did not meliorate the decline of sexual behavior. Neonatal MSG treatment also induced reductions of body weight in male rats, higher diurnal percentages of food and water intake in male rats than in female rats, and a decline of water-to-food ratio. These observations suggest that neonatal male rats show higher susceptibility to glutamate-induced dysfunction of sexual and ingestive behaviors than females. Our findings also suggest a sex difference in the mechanism of dysfunction of sexual behavior, i.e., decreased copulatory activity in male rats is mainly due to CNS damage, in contrast to decreased blood progesterone level in female rats.

This study elucidated the stage-specific roles of FGF2 signaling during neural development using in-vitro human embryonic stem cell-based developmental modeling. We found that the dysregulation of FGF2 signaling prior to the onset of neural induction resulted in the malformation of neural rosettes (a neural tube-like structure), despite cells having undergone neural induction. The aberrant neural rosette formation may be attributed to the misplacement of ZO-1, which is a polarized tight junction protein and shown co-localized with FGF2/FGFR1 in the apical region of neural rosettes, subsequently led to abnormal neurogenesis. Moreover, the FGF2 signaling inhibition at the stage of neural rosettes caused a reduction in cell proliferation, an increase in numbers of cells with cell-cycle exit, and premature neurogenesis. These effects may be mediated by NUMB, to which expression was observed enriched in the apical region of neural rosettes after FGF2 signaling inhibition coinciding with the disappearance of PAX6+/Ki67+ neural stem cells and the emergence of MAP2+ neurons. Moreover, our results suggested that the hESC-based developmental system reserved a similar neural stem cell niche in vivo.