Expression of disrupted in schizophrenia 1 (DISC1) protein in the adult and developing mouse brain indicates its role in neurodevelopment.

Disrupted in Schizophrenia 1 (DISC1) was identified as a potential susceptibility gene for schizophrenia due to its disruption by a balanced t(1;11) (q42;q14) translocation, which has been shown to cosegregate with major psychiatric disease in a large Scottish family. We have recently presented evidence that DISC1 exists in a neurodevelopmentally regulated protein complex with Nudel. In this study, we report the protein expression profile of DISC1 in the adult and developing mouse brain utilizing immunohistochemistry and quantitative Western blot. In the adult mouse brain, DISC1 is expressed in neurons within various brain areas including the olfactory bulb, cortex, hippocampus, hypothalamus, cerebellum and brain stem. During development, DISC1 protein is detected at all stages, from E10 to 6 months old, with two significant peaks of protein expression of a DISC1 isoform at E13.5 and P35. Interestingly, these time points correspond to critical stages during mouse development, the active neurogenesis period in the developing brain and the period of puberty. Together, these results suggest that DISC1 may play a critical role in brain development, consistent with the neurodevelopmental hypothesis of the etiology of schizophrenia.

Glutamatergic induction of CREB phosphorylation and Fos expression in primary cultures of the suprachiasmatic hypothalamus in vitro is mediated by co-ordinate activity of NMDA and non-NMDA receptors.

Exposure of Syrian hamsters to light 1 h after lights-off rapidly (10 min) induced nuclear immunoreactivity (-ir) to the phospho-Ser133 form of the Ca2+/cAMP response element (CRE) binding protein (pCREB) in the retinorecipient zone of the suprachiasmatic nuclei (SCN). Light also induced nuclear Fos-ir in the same region of the SCN after 1 h. The glutamatergic N-methyl-D-aspartate (NMDA) receptor blocker MK801 attenuated the photic induction of both factors. To investigate glutamatergic regulation of pCREB and Fos further, tissue blocks and primary cultures of neonatal hamster SCN were examined by Western blotting and immunocytochemistry in vitro. On Western blots of SCN tissue, the pCREB-ir signal at 45 kDa was enhanced by glutamate or a mixture of glutamatergic agonists (NMDA, amino-methyl proprionic acid (AMPA), and Kainate (KA)), whereas total CREB did not change. Glutamate or the mixture of agonists also induced a 56 kDa band identified as Fos protein in SCN tissue. In dissociated cultures of SCN, glutamate caused a rapid (15 min) induction of nuclear pCREB-ir and Fos-ir (after 60 min) exclusively in neurones, both GABA-ir and others. Treatment with NMDA alone had no effect on pCREB-ir. AMPA alone caused a slight increase in pCREB-ir. However, kainate alone or in combination with NMDA and AMPA induced nuclear pCREB-ir equal to that induced by glutamate. The effects of glutamate on pCREB-ir and Fos-ir were blocked by antagonists of both NMDA (MK801) and AMPA/KA (NBQX) receptors. In the absence of extracellular Mg2+, MK801 blocked glutamatergic induction of Fos-ir. However, the AMPA/KA receptor antagonist was no longer effective at blocking glutamatergic induction of either Fos-ir or pCREB-ir, consistent with the model that glutamate regulates gene expression in the SCN by a co-ordinate action through both NMDA and AMPA/KA receptors. Glutamatergic induction of nuclear pCREB-ir in GABA-ir neurones was blocked by KN-62 an inhibitor of Ca2+/Calmodulin (CaM)-dependent kinases, implicating Ca2+-dependent signalling pathways in the glutamatergic regulation of gene expression in the SCN.

Stimuli which entrain the circadian clock of the neonatal Syrian hamster in vivo regulate the phosphorylation of the transcription factor CREB in the suprachiasmatic nucleus in vitro.

Photic resetting of the adult mammalian circadian clock in vivo is associated with phosphorylation of the Ser133 residue of the calcium/cyclic AMP response-element binding-protein (CREB) in the retinorecipient region of the suprachiasmatic nucleus (SCN). Western blotting and immunocytochemistry were used to investigate whether agonists known to reset the clock of neonatal hamsters in vivo are also able to influence the phosphorylation of CREB in the suprachiasmatic hypothalamus in vitro. Antisera raised against synthetic CREB peptide sequences were used to differentiate between total CREB and the Ser133 phosphorylated form of CREB (pCREB). Western blot analysis of proteins isolated from suprachiasmatic tissue of 1-day-old Syrian hamsters revealed bands at approximately 45 kDa corresponding to total CREB and pCREB. Treatment of the tissue with a mixture of glutamatergic agonists [N-methyl-D-aspartate (NMDA), amino-methyl proprionic acid (AMPA) and kainate, all at 1 microM], or native glutamate (1 microM) had no effect on the total CREB signal, but increased the pCREB signal, indicative of agonist-stimulated phosphorylation of CREB on Ser133. A similar effect was seen following treatment of the suprachiasmatic blocks with either dopamine (1 microM) or forskolin (1 microM). Simultaneous treatment with melatonin (1 microM) significantly attenuated stimulation by forskolin. The effect of the agonists on nuclear pCREB-immunoreactivity (-ir) was investigated in primary cultures which contained a mixture of cell types characteristic of the suprachiasmatic nuclei in vivo. Basal expression of nuclear total CREB-ir was high, whereas expression of pCREB-ir was low. Treatment with glutamate (1 microM) or dopamine (1 microM) had no effect on total CREB-ir, but increased pCREB-ir in approximately 50 and 30% of cells, respectively, whereas forskolin (1 microM) increased pCREB-ir in almost all cells (> 90%). The effects of all three agonists were rapid (< 15 min), and dose and time dependent. Melatonin reversed the effects of forskolin in mixed cultures, but not in pure astrocyte cultures. Dual-immunocytochemistry (ICC) revealed that glutamate (1 microM) increased nuclear pCREB-ir in cells immunoreactive for microtubule-associated protein II (MAP II-ir), but not other cells, indicating an effect predominantly on neurons. This occurred equally in gamma-amino butyric acid (GABA)-ir and non-GABA-ir neurons. Dopamine (1 microM) was more selective, increasing pCREB-ir only in GABA-ir neurons, whereas forskolin increased pCREB-ir in all cells. The specific stimulation of pCREB-ir in GABA-ir neurons by dopamine was reversed by melatonin, but melatonin had no effect on the increase in pCREB-ir induced in GABA-ir neurons by glutamate. These results demonstrate that agonists known to entrain the circadian clock in vivo modulate phosphorylation of CREB in GABA-ir neurons derived from the neonatal suprachiasmatic nuclei.

Melatonin-sensitive, serum-stimulated signalling in ovine pars tuberalis.

In primary cultures of ovine pars tuberalis (oPT), serum acts through melatonin-sensitive mechanisms independent of cyclic AMP to increase the phosphorylation of the Ca2+/cyclic AMP response element binding protein (CREB). Immunocytochemical and biochemical assays were used to characterize the active components of serum and the signalling pathways through which they and melatonin function in oPT. The stimulatory effect of serum was heat-labile, sensitive to precipitation by methanol, and required components with a mass greater than 10 KDa implicating peptide or protein factors as the active agent. Serum increased the cytosolic free Ca2+ concentration ([Ca2+]i) of oPT cells. Serum also enhanced the release of [3H]-choline and [3H]-arachidonic acid from prelabeled cells, demonstrating that factors present in serum increase the breakdown of cellular phospholipids. This effect, however, was not blocked by melatonin (1 microM). Serum also caused a dose-dependent increase in levels of immediate early gene immunoreactivity, confirming that factors in serum have the ability to control transcription in the oPT. Down-regulation of protein kinase C (PKC) by treatment with 12-0-tetradecanoylphorbol-13-acetate (TPA, 100 nM) or treatment with a specific PKC inhibitor (RO-31-8220, 1 microM), did not affect protein kinase A-mediated stimulation of CREB phosphorylation. However, down-regulation of PKC blocked the acute stimulatory effects of TPA (100 nM) and of serum (1%). Moreover, RO-31-8220 abolished the stimulatory effect of TPA (100 nM) and strongly attenuated that of serum (1%). These results demonstrate that serum increases the phosphorylation of CREB by stimulating cyclic AMP-independent, PKC-dependent, signalling pathways within the oPT. PKC may be activated through increased phospholipid catabolism and/or raised [Ca2+]i.

The inhibitory action of melatonin in the ovine pars tuberalis is not dependent on changes in plasma membrane potential.

Treatment of ovine pars tuberalis (oPT) cultures with forskolin activates adenylyl cyclase, leading to increased levels of cyclic AMP, activation of protein kinase A, phosphorylation of the calcium/cyclic AMP response-element binding protein and the increased synthesis and secretion of several proteins. Simultaneous treatment with melatonin inhibits or reverses these effects of forskolin. In the neonatal rat pituitary, the inhibitory effects of melatonin are mediated by changes in membrane potential. This study therefore investigated whether the inhibitory action of melatonin in oPT cultures is also dependent on the modulation of plasma membrane potential. Treatment of cultures with the ionophore valinomycin selectively permeabilised the cell plasma membrane to potassium, thereby causing membrane hyperpolarisation. In cultures of oPT, valinomycin inhibited in a concentration-dependent manner (maximal effect 2 microM) the stimulatory action of forskolin (1 microM) on intracellular levels of cyclic AMP, indicating that the activity of adenylyl cyclase in this tissue is sensitive to hyperpolarisation of the plasma membrane. However, increasing the extracellular concentration of potassium from 5 mM to 100 mM, which would depolarise the plasma membrane, had no effect on the inhibitory action of melatonin (1 microM) in forskolin-stimulated cultures. This indicated that melatonin could be effective in cells with sustained depolarisation. To test directly whether integrity of the plasma membrane is essential for melatonin to inhibit adenylyl cyclase, cultures were treated with the cholesterol-chelating agent saponin (50 micrograms/ml). Saponin increased cellular permeability to trypan blue and enhanced the release of the cytoplasmic enzyme lactate dehydrogenase to the extracellular medium, demonstrating that cell plasma membranes had been permeabilised, thereby abolishing membrane polarity.(ABSTRACT TRUNCATED AT 250 WORDS)