Sivan Subburaju

Regulation of the GABA cell phenotype in hippocampus of schizophrenics and bipolars

GABAergic dysfunction is present in the hippocampus in schizophrenia (SZ) and bipolar disorder (BD). The trisynaptic pathway was “deconstructed” into various layers of sectors CA3/2 and CA1 and gene expression profiling performed. Network association analysis was used to uncover genes that may be related to regulation of glutamate decarboxylase 67 (GAD67), a marker for this system that has been found by many studies to show decreased expression in SZs and BDs. The most striking change was a down-regulation of GAD67 in the stratum oriens (SO) of CA2/3 in both groups; CA1 only showed changes in the SO of schizophrenics. The network generated for GAD67 contained 25 genes involved in the regulation of kainate receptors, TGF-β and Wnt signaling, as well as transcription factors involved in cell growth and differentiation. In SZs, IL-1β, (GRIK2/3), TGF-β2, TGF-βR1, histone deacetylase 1 (HDAC1), death associated protein (DAXX), and cyclin D2 (CCND2) were all significantly up-regulated, whereas in BDs, PAX5, Runx2, LEF1, TLE1, and CCND2 were significantly down-regulated. In the SO of CA1 of BDs, where GAD67 showed no expression change, TGF-β and Wnt signaling genes were all up-regulated, but other transcription factors showed no change in expression. In other layers/sectors, BDs showed no expression changes in these GAD67 network genes. Overall, these results are consistent with the hypothesis that decreased expression of GAD67 may be associated with an epigenetic mechanism in SZ. In BD, however, a suppression of transcription factors involved in cell differentiation may contribute to GABA dysfunction.

The Parvocellular Vasopressinergic System and Responsiveness of the Hypothalamic Pituitary Adrenal Axis during Chronic Stress

Vasopressin (VP) secreted from parvocellular neurons of the hypothalamic paraventricular nucleus (PVN) stimulates pituitary adrenocorticotropic hormone (ACTH) secretion, through interaction with receptors of the V1b subtype (V1bR) in the pituitary corticotroph, mainly by potentiating the stimulatory effects of corticotrophin-releasing hormone (CRH). Chronic stress paradigms associated with corticotroph hyperresponsiveness lead to preferential expression of hypothalamic VP over CRH and upregulation of pituitary V1bR, suggesting that VP has a primary role during adaptation of the hypothalamic pituitary adrenal (HPA) axis to long-term stimulation. However, studies using pharmacological or genetic ablation of V1bR have shown that VP is required for full ACTH responses to some stressors, but not for the sensitization of ACTH responses to a novel stress observed during chronic stress. Studies using minipump infusion of a peptide V1 antagonist in long-term adrenalectomized rats have revealed that VP mediates proliferative responses in the pituitary. Nevertheless, only a minor proportion of cells undergoing mitogenesis co-express markers for differentiated corticotrophs or precursors, suggesting that new corticotrophs are recruited from yet undifferentiated cells. The overall evidence supports a limited role of VP regulating acute ACTH responses to some acute stressors and points to cell proliferation and pituitary remodelling as alternative roles for the marked increases in parvocellular vasopressinergic activity during prolonged activation of the HPA axis.

Formation of the retinotectal projection requires Esrom, an ortholog of PAM (protein associated with Myc)

Visual system development is dependent on correct interpretation of cues that direct growth cone migration and axon branching. Mutations in the zebrafish esrom gene disrupt bundling and targeting of retinal axons, and also cause ectopic arborization. By positional cloning, we establish that esrom encodes a very large protein orthologous to PAM (protein associated with Myc)/Highwire/RPM-1. Unlike motoneurons in Drosophila highwire mutants, retinal axons in esrom mutants do not arborize excessively, indicating that Esrom has different functions in the vertebrate visual system. We show here that Esrom has E3 ligase activity and modulates the amount of phosphorylated Tuberin, a tumor suppressor, in growth cones. These data identify a mediator of signal transduction in retinal growth cones, which is required for topographic map formation.

Interaction Between Oestrogen and Oxytocin on Hypothalamic‐Pituitary‐Adrenal Axis Activity

In addition to its role in reproduction, oxytocin has central actions modulating behavioural and hypothalamic‐pituitary‐adrenal (HPA) axis responses during late pregnancy and lactation. The hypothesis that ovarian hormones modulate the effects of oxytocin on HPA axis activity was studied in 7‐day ovariectomised rats receiving oestradiol with or without progesterone replacement and intracerebroventricular (i.c.v) minipump infusion of oxytocin (100 ng/h). In an initial experiment, i.c.v. oxytocin had no effect on basal or restraint‐stimulated plasma adrenocorticotrophic hormone (ACTH) and corticosterone concentrations or hypothalamic corticotrophin‐releasing factor (CRF) mRNA expression with low oestradiol replacement alone but it had a stimulatory effect in the presence of low oestradiol and progesterone. To investigate further whether oestradiol modulates central actions of oxytocin, rats received low dioestrous (low), pro‐oestrous (medium) or pregnancy (high) oestradiol replacement levels, yielding plasma concentrations of < 5, 17.3 ± 4.5 and 258 ± 32 pg/ml, respectively, with or without i.c.v. oxytocin. Oestradiol caused dose‐dependent increases in basal plasma ACTH and corticosterone concentrations but decreased the ACTH response to restraint stress. In parallel to the changes in basal plasma ACTH, high oestrogen increased basal CRF hnRNA, CRF mRNA in the paraventricular nucleus and pro‐opiomelanocortin (POMC) mRNA in the pituitary gland, while decreasing restraint stress‐stimulated levels. Intracerebroventricular administration of oxytocin reduced basal and stress‐stimulated plasma ACTH, hypothalamic CRF hnRNA (30 min), CRF mRNA and pituitary POMC mRNA (4 h) levels parallel to the increases induced by elevating plasma oestradiol. The present study demonstrates the converse effects of oestradiol on basal and restraint stress‐stimulated basal HPA axis activity, and that the ability of central oxytocin to inhibit HPA axis activity depends on the levels of circulating oestradiol.

Circuitry-based gene expression profiles in GABA cells of the trisynaptic pathway in schizophrenics versus bipolars

Significant reductions in GABAergic cell numbers and/or activity have been demonstrated in the hippocampus of subjects with schizophrenia and bipolar disorder. To understand how different subpopulations of interneurons are regulated, laser microdissection and gene expression profiling have been used to “deconstruct” the trisynaptic pathway, so that subtypes of GABA cells could be defined by their location in various layers of CA3/2 and CA1. The results suggest that the cellular endophenotypes for SZ and BD may be determined by multiple factors that include unique susceptibility genes for the respective disorders and altered integration among hippocampal GABA cells with extrinsic and intrinsic afferent fiber systems. The extensive and intricate data that has come from this study has provided insights into how a complex circuit, like the trisynaptic pathway, may be regulated in human hippocampus in both health and disease.

Circuit- and Diagnosis-Specific DNA Methylation Changes at γ-Aminobutyric Acid-Related Genes in Postmortem Human Hippocampus in Schizophrenia and Bipolar Disorder

IMPORTANCE Dysfunction related to γ-aminobutyric acid (GABA)-ergic neurotransmission in the pathophysiology of major psychosis has been well established by the work of multiple groups across several decades, including the widely replicated downregulation of GAD1. Prior gene expression and network analyses within the human hippocampus implicate a broader network of genes, termed the GAD1 regulatory network, in regulation of GAD1 expression. Several genes within this GAD1 regulatory network show diagnosis- and sector-specific expression changes within the circuitry of the hippocampus, influencing abnormal GAD1 expression in schizophrenia and bipolar disorder. OBJECTIVE To investigate the hypothesis that aberrant DNA methylation contributes to circuit- and diagnosis-specific abnormal expression of GAD1 regulatory network genes in psychotic illness. DESIGN, SETTING, AND PARTICIPANTS This epigenetic association study targeting GAD1 regulatory network genes was conducted between July 1, 2012, and June 30, 2014. Postmortem human hippocampus tissue samples were obtained from 8 patients with schizophrenia, 8 patients with bipolar disorder, and 8 healthy control participants matched for age, sex, postmortem interval, and other potential confounds from the Harvard Brain Tissue Resource Center, McLean Hospital, Belmont, Massachusetts. We extracted DNA from laser-microdissected stratum oriens tissue of cornu ammonis 2/3 (CA2/3) and CA1 postmortem human hippocampus, bisulfite modified it, and assessed it with the Infinium HumanMethylation450 BeadChip (Illumina, Inc). The subset of CpG loci associated with GAD1 regulatory network genes was analyzed in R version 3.1.0 software (R Foundation) using the minfi package. Findings were validated using bisulfite pyrosequencing. MAIN OUTCOMES AND MEASURES Methylation levels at 1308 GAD1 regulatory network-associated CpG loci were assessed both as individual sites to identify differentially methylated positions and by sharing information among colocalized probes to identify differentially methylated regions. RESULTS A total of 146 differentially methylated positions with a false detection rate lower than 0.05 were identified across all 6 groups (2 circuit locations in each of 3 diagnostic categories), and 54 differentially methylated regions with P < .01 were identified in single-group comparisons. Methylation changes were enriched in MSX1, CCND2, and DAXX at specific loci within the hippocampus of patients with schizophrenia and bipolar disorder. CONCLUSIONS AND RELEVANCE This work demonstrates diagnosis- and circuit-specific DNA methylation changes at a subset of GAD1 regulatory network genes in the human hippocampus in schizophrenia and bipolar disorder. These genes participate in chromatin regulation and cell cycle control, supporting the concept that the established GABAergic dysfunction in these disorders is related to disruption of GABAergic interneuron physiology at specific circuit locations within the human hippocampus.

Site-specific regulation of cell cycle and DNA repair in post-mitotic GABA cells in schizophrenic versus bipolars.

GABA cell dysfunction in both schizophrenia (SZ) and bipolar disorder (BD) involves decreased GAD67 expression, although this change involves fundamentally different networks of genes in the 2 disorders. One gene that is common to these 2 networks is cyclin D2, a key component of cell cycle regulation that shows increased expression in SZ, but decreased expression in BD. Because of the importance of cell cycle regulation in maintaining functional differentiation and DNA repair, the current study has examined the genes involved in the G1 and G2 checkpoints to generate new hypotheses regarding the regulation of the GABA cell phenotype in the hippocampus of SZ and BD. The results have demonstrated significant changes in cell cycle regulation in both SZ and BD and these changes include the transcriptional complex (TC) that controls the expression of E2F/DP-1 target genes critical for progression to G2/M. The methyl-CpG binding domain protein (MBD4) that is pivotal for DNA repair, is significantly up-regulated in the stratum oriens (SO) of CA3/2 and CA1 in SZs and BDs. However, other genes associated with the TC, and the G1 and G2 checkpoints, show complex changes in expression in the SO of CA3/2 and CA1 of both SZs and BDS. Overall, the patterns of expression observed have suggested that the regulation of functional differentiation and/or genomic integrity of hippocampal GABA cells varies according to diagnosis and their location within the trisynaptic pathway.

Prolactin Activates Mitogen-Activated Protein Kinase Signaling and Corticotropin Releasing Hormone Transcription in Rat Hypothalamic Neurons

Prolactin (PRL) modulates maternal behavior and mediates hypothalamic pituitary adrenal axis inhibition during lactation via PRL receptors in the brain. To identify mechanisms mediating these effects, we examined the effects of PRL on signaling and CRH transcription in hypothalamic neurons in vivo and in vitro. Western blot of hypothalamic proteins from rats receiving intracerebroventricular PRL injection revealed increases in phosphorylation of the MAPK and ERK. Double-staining immunohistochemistry demonstrated phosphorylated ERK localization in parvocellular CRH neurons as well as magnocellular vasopressin and oxytocin neurons of the hypothalamic paraventricular (PVN) and supraoptic nuclei. PRL also induced ERK phosphorylation in vitro in the hypothalamic cell line, 4B, which expresses PRL receptors, and in primary hypothalamic neuronal cultures. Using reporter gene assays in 4B cells, or quantitative RT-PCR for primary transcript in hypothalamic cell cultures, PRL potentiated forskolin-stimulated CRH transcription through activation of the ERK/MAPK pathway. The effect of PRL in hypothalamic cell cultures was unaffected by tetrodotoxin, suggesting a direct effect on CRH neurons. The data show that PRL activates the ERK/MAPK pathway and facilitates CRH transcription in CRH neurons, suggesting that the inhibitory effect of PRL on hypothalamo-pituitary-adrenal axis activity reported in vivo is indirect and probably mediated through modulation of afferent pathways to the PVN. In addition, the prominent stimulatory action of PRL on the ERK/MAPK pathway in the hypothalamic PVN and supraoptic nucleus is likely to mediate neuroplasticity of the neuroendocrine system during lactation.

Vasopressin Does Not Mediate Hypersensitivity of the Hypothalamic Pituitary Adrenal Axis during Chronic Stress

The hypothesis that vasopressin (VP) becomes the main mediator of pituitary corticotroph responsiveness during chronic hypothalamic pituitary adrenal (HPA) axis activation was tested by examining the effect of pharmacologic VP receptor blockade on the adrenocorticotropic hormone (ACTH) and corticosterone responses of 14‐day repeatedly restrained rats. In spite of the increased vasopressinergic activity, repeatedly restrained rats showed lower ACTH and corticosterone responses to 10 min white noise compared with handled controls. These responses were unchanged by injection of the nonpeptide‐selective V1b receptor antagonist SSR149415 i.v., 1 h before noise application. In contrast to noise stress, plasma ACTH responses to i.p. hypertonic saline injection were enhanced in the repeatedly restrained rats compared with handled controls, but responses were also unaffected by SSR149415 administered orally, daily 1 h before restraint. Since SSR149415 effectiveness was low, we used minipump infusion of the peptide V1 receptor antagonist, dGly[Phaa1,D‐tyr(et), Lys, Arg]VP (V1‐Ant) for 14 days, which effectively blocked ACTH responses to exogenous VP. Chronic V1‐Ant infusion reduced plasma ACTH responses to i.p. hypertonic saline in handled controls but not in repeatedly restrained rats. These data suggest that the increased vasopressinergic activity characteristic of chronic stress plays roles other than mediating the hypersensitivity of the HPA axis to a novel stress.

Inhibition of vasopressin V1b receptor translation by upstream open reading frames in the 5'-untranslated region.

The 5′‐UTR of the vasopressin V1b receptor (V1bR) mRNA contains small open reading frames (ORF) located upstream (u) of the main ORF encoding the V1bR. The ability of the three proximal uORFs to be translated into peptides and their influence on V1bR translation was examined using fusion constructs of uORFs and V5 epitope, or ATG/ATA uORF mutations in the V1bR cDNA. In vitro translation and western blot analysis after transfection of uORF1‐V5 or uORF2‐V5 into cells revealed that uORF1 can be translated. As predicted by computer analysis, in vitro translation using a rabbit reticulocyte/canine microsome system, immunohistochemistry and western blot in membranes of transfected cells with uORF1‐V5 revealed translocation of the uORF1 peptide into membrane fractions. In vitro translation of V1bR cDNA with mutations of the two uORFs proximal to the initiating methionine, uORFs 1 and 2 (Mut 1–2), or uORF2 (Mut 2) showed significantly increased translation of a 46 kDa band corresponding to the V1bR, compared with wild‐type (WT) V1bR, an effect that was attenuated by cotranslation of uORF1‐V5. Consistently, VP‐induced inositol phosphate formation was higher in Chinese hamster ovay cells transfected with Mut 1–2 than with WT V1bR. Immunohistochemical and western blot analysis, using an antibody against uORF1, revealed peptide immunoreactivity in rat pituitary but not in liver. Pituitary uORF immunoreactivity increased following glucocorticoid administration. The present study shows that uORFs in the 5′‐UTR of the V1bR mRNA inhibit V1bR translation, and suggests that translation of a 38‐amino acid membrane peptide encoded by uORF1 exerts tonic inhibition of V1bR translation.