17ß-estradiol-induced regulation of the novel 5-HT1A-related transcription factors NUDR and Freud-1 in SH SY5Y cells.

Nuclear deformed epidermal autoregulatory factor-1 (NUDR/Deaf-1) and five prime repressor element under dual repression (Freud-1) are novel transcriptional regulators of the 5-HT(1A) receptor, a receptor that has been implicated in the pathophysiology of various psychiatric illnesses. The antidepressant effect of 17ß-Estradiol (17ßE(2)) is purported to involve the downregulation of this receptor. We investigated the possible role of NUDR and Freud-1 in 17ßE(2)-induced downregulation of the 5-HT(1A) receptor in the neuroblastoma cell line SH SY5Y. Cells were treated with 10 nM of 17ßE(2) for 3 or 48 h, followed by a 24-h withdrawal period. Proteins were isolated and analyzed by western blotting. 17ßE(2) treatment increased NUDR immunoreactivity while Freud-1 and the 5-HT(1A) receptor showed significant decreases. Upon withdrawal of 17ßE(2), protein expression returned to control levels, except for NUDR, which remained significantly elevated in the 3-h treatment. Taken together, these data support a non-genomic downregulation of 5-HT(1A) receptor protein by 17ßE(2), which does not involve NUDR and Freud-1. Rather, changes in both transcription factors seem to be compensatory/homeostatic responses to changes in 5-HT(1A) receptor induced by 17ßE(2). These observations further highlight the importance of NUDR and Freud-1 in regulating 5-HT(1A) receptor expression.

Agmatine protects against cell damage induced by NMDA and glutamate in cultured hippocampal neurons.

Agmatine is a polyamine and has been considered as a novel neurotransmitter or neuromodulator in the central nervous system. In the present study, the neuroprotective effect of agmatine against cell damage caused by N-methyl-D-aspartate (NMDA) and glutamate was investigated in cultured rat hippocampal neurons. Lactate dehydrogenase (LDH) activity assay, beta-tubulin III immunocytochemical staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL) assay were conducted to detect cell damage. Exposure of 12-day neuronal cultures of rat hippocampus to NMDA or glutamate for 1 h caused a concentration-dependent neurotoxicity, as indicated by the significant increase in released LDH activities. Addition of 100 microM agmatine into media ablated the neurotoxicity induced by NMDA or glutamate, an effect also produced by the specific NMDA receptor antagonist dizocilpine hydrogen maleate (MK801). Arcaine, an analog of agmatine with similar structure as agmatine, fully prevented the NMDA- or glutamate-induced neuronal damage. Spermine and putrescine, the endogenous polyamine and metabolic products of agmatine without the guanidine moiety of agmatine, failed to show this effect, indicating a structural relevance for this neuroprotection. Immunocytochemical staining and TUNEL assay confirmed the findings in the LDH measurement. That is, agmatine and MK801 markedly attenuated NMDA-induced neuronal death and significantly reduced TUNEL-positive cell numbers induced by exposure of cultured hippocampal neurons to NMDA. Taken together, these results demonstrate that agmatine can protect cultured hippocampal neurons from NMDA- or glutamate-induced excitotoxicity, through a possible blockade of the NMDA receptor channels or a potential anti-apoptotic property.

Oligodendrocyte morphometry and expression of myelin - Related mRNA in ventral prefrontal white matter in major depressive disorder.

White matter disturbance in the ventral prefrontal cortex (vPFC) in major depressive disorder (MDD) has been noted with diffusion tensor imaging (DTI). However, the cellular and molecular pathology of prefrontal white matter in MDD and potential influence of antidepressant medications is not fully understood. Oligodendrocyte morphometry and myelin-related mRNA and protein expression was examined in the white matter of the vPFC in MDD. Sections of deep and gyral white matter from the vPFC were collected from 20 subjects with MDD and 16 control subjects. Density and size of CNPase-immunoreactive (-IR) oligodendrocytes were estimated using 3-dimensional cell counting. While neither density nor soma size of oligodendrocytes was significantly affected in deep white matter, soma size was significantly decreased in the gyral white matter in MDD. In rhesus monkeys treated chronically with fluoxetine there was no significant effect on oligodendrocyte morphometry. Using quantitative RT-PCR to measure oligodendrocyte-related mRNA for CNPase, PLP1, MBP, MOG, MOBP, Olig1 and Olig2, in MDD there was a significantly reduced expression of PLP1 mRNA (which positively correlated with smaller sizes) and increased expression of mRNA for CNPase, OLIG1 and MOG. The expression of CNPase protein was significantly decreased in MDD. Altered expression of four myelin genes and CNPase protein suggests a mechanism for the degeneration of cortical axons and dysfunctional maturation of oligodendrocytes in MDD. The change in oligodendrocyte morphology in gyral white matter may parallel altered axonal integrity as revealed by DTI.

Reduced phosphorylation of the mTOR signaling pathway components in the amygdala of rats exposed to chronic stress.

The activity of the mammalian target of rapamycin (mTOR), an ubiquitously expressed serine/threonine kinase, is central to the regulation of translation initiation and, consequently protein synthesis required for long-term potentiation and new synaptic connections. Recent studies show that activation of the mTOR signaling pathway is required for the rapid antidepressant actions of glutamate N-methyl-d-aspartate (NMDA) receptor antagonists such as ketamine. Our prior work documented the first evidence of robust deficits in the mTOR signaling pathway in the prefrontal cortex (PFC) from subjects diagnosed with major depressive disorder (MDD). The goal of this study was to determine whether alterations in mTOR signaling can be observed in rats exposed to the chronic unpredictable stress (CUS) model of depression. In the present study, we examined the effect of CUS on the expression of phosphorylated mTOR and its downstream signaling components in the frontal cortex, hippocampus, amygdala, and dorsal raphe. We also examined the effect of CUS on the expression of kinases that phosphorylate mTOR such as extracellular signal-regulated kinase (ERK1/2) and protein kinase B/Akt (Akt1). In addition, we examined the effect of stress on the phosphorylation of GluR1 an, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit. We found that eight-weeks of CUS exposure significantly decreased the phosphorylation levels of mTOR and its downstream signaling components in the amygdala. Reduced level of phospho-mTOR in the amygdala was accompanied by decreased phosphorylation of ERK-1/2, Akt-1, and GluR1. No significant changes were seen in the frontal cortex, hippocampus, or dorsal raphe. Our study demonstrates that long-term stress exposure results in brain region-specific abnormalities in signaling pathways previously linked to novel mechanisms for rapid antidepressant effects. These observations are in line with evidence showing that mTOR and its upstream and downstream signaling partners could be important targets for the development of novel antidepressants.

Gene expression analysis of novel genes in the prefrontal cortex of major depressive disorder subjects.

Dysregulation of the glutamatergic system has been implicated not only in the treatment of major depressive disorder (MDD), but also in the excitotoxic effects of stress and anxiety on the prefrontal cortex, which may precede the onset of a depressive episode. Our previous studies demonstrate marked deficits in prominent postsynaptic proteins involved in glutamate neurotransmission in the prefrontal cortex (PFC), Brodmann's area 10 (BA 10) from subjects diagnosed with major depressive disorder (MDD). In the same group of subjects we have identified deficits in expression and phosphorylation level of key components of the mammalian target of rapamycin (mTOR) signaling pathway, known to regulate translation initiation. Based on our previous findings, we have postulated that glutamate-dependent dysregulation of mTOR-initiated protein synthesis in the PFC may underlie the pathology of MDD. The aim of this study was to use the NanoString nCounter System to perform analysis of genes coding for glutamate transporters, glutamate metabolizing enzymes, neurotrophic factors and other intracellular signaling markers involved in glutamate signaling that were not previously investigated by our group in the PFC BA 10 from subjects with MDD. We have analyzed a total of 200 genes from 16 subjects with MDD and 16 healthy controls. These are part of the same cohort used in our previous studies. Setting our cutoff p-value≤0.01, marked upregulation of genes coding for mitochondrial glutamate carrier (GC1; p=0.0015), neuropilin 1 (NRP-1; p=0.0019), glutamate receptor ionotropic N-methyl-d-aspartate-associated protein 1 (GRINA; p=0.0060), and fibroblast growth factor receptor 1 (FGFR-1; p=0.010) was identified. No significant differences in expression of the remaining 196 genes were observed between MDD subjects and controls. While upregulation of FGFR-1 has been previously shown in MDD; abnormalities in GC-1, GRINA, and NRP-1 have not been reported. Therefore, this postmortem study identifies GC1, GRINA, and NRP-1 as novel factors associated with MDD; however, future studies will be needed to address the significance of these genes in the pathophysiology of depression and antidepressant activity.

The mTOR signaling pathway in the prefrontal cortex is compromised in major depressive disorder.

Recent studies demonstrate that rapid antidepressant response to ketamine is mediated by activation of the mammalian target of rapamycin (mTOR) signaling pathway, leading to increased synaptic proteins in the prefrontal cortex (PFC) of rats. Our postmortem studies indicate robust deficits in prominent postsynaptic proteins including N-methyl-d-aspartate (NMDA) receptor subunits (NR2A, NR2B), metabotropic glutamate receptor subtype 5 (mGluR5) and postsynaptic density protein 95kDa (PSD-95) in the PFC in major depressive disorder (MDD). We hypothesize that deficits in the mTOR-dependent translation initiation pathway contribute to the molecular pathology seen in the PFC of MDD subjects, and that a rapid reversal of these abnormalities may underlie antidepressant activity. The majority of known translational regulation occurs at the level of initiation. mTOR regulates translation initiation via its downstream components: p70-kDa ribosomal protein S6 kinase (p70S6K), and eukaryotic initiation factors 4E and 4B (eIF4E and eIF4B). In this study, we examined the expression of mTOR and its core downstream signaling targets: p70S6K, eIF4E, and eIF4B in the PFC of 12 depressed subjects and 12 psychiatrically healthy controls using Western blot. Levels of eIF4E phosphorylated at serine 209 (p-eIF4E-Ser209) and eIF4B phosphorylated at serine 504 (p-eIF4B-Ser504) were also examined. Adjacent cortical tissue samples from both cohorts of subjects were used in our previous postmortem analyses. There was a significant reduction in mTOR, p70S6K, eIF4B and p-eIF4B protein expression in MDD subjects relative to controls. No group differences were observed in eIF4E, p-eIF4E or actin levels. Our findings show deficits in mTOR-dependent translation initiation in MDD particularly via the p70S6K/eIF4B pathway, and indicate a potential association between marked deficits in synaptic proteins and dysregulation of mTOR signaling in MDD.

Ethanol increases TIEG2-MAO B cell death cascade in the prefrontal cortex of ethanol-preferring rats.

Brain cell loss has been reported in subjects with alcoholism. However, the molecular mechanisms are unclear. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and monoamine oxidase B (MAO B) reportedly play a role in cellular dysfunction with regards to ethanol exposure. We have recently reported that GAPDH protein expression was increased in the brains of rats fed with ethanol. Furthermore, GAPDH interacts with the transcriptional activator, transforming growth factor-beta-inducible early gene 2 (TIEG2), to augment TIEG2-mediated MAO B activation, resulting in neuronal cell damage due to ethanol exposure. The current study investigates whether the TIEG2-MAO B cascade is also active in the brains of rats fed with ethanol. Ten ethanol-preferring rats were fed with a liquid diet containing ethanol, with increasing amounts of ethanol up to a final concentration of 6.4% representing a final diet containing 36% of calories for 28 days. Ten control rats were fed the liquid diet without ethanol. The expression of TIEG2 protein, MAO B mRNA levels, MAO B catalytic activity, and the levels of anti-apoptotic protein Bcl 2 and apoptotic protein caspase 3 were determined in the prefrontal cortex of the rats. Ethanol significantly increased protein levels of TIEG2, active caspase 3, MAO B mRNA and enzyme activity, but significantly decreased Bcl 2 protein expression compared to control rats. In summary, ethanol increases the TIEG2-MAO B brain cell death cascade in rat brains, suggesting that the TIEG2-MAO B pathway is a novel pathway for brain cell damage resulting from ethanol exposure, and may contribute to chronic alcohol-induced brain damage.

Chronic social defeat downregulates the 5-HT1A receptor but not Freud-1 or NUDR in the rat prefrontal cortex.

The serotonin 1A receptor (5-HT1A) and its associated transcriptional regulators, five prime repressor element under dual repression (Freud-1) and nuclear-deformed epidermal autoregulatory factor (NUDR/Deaf-1) have been previously found to be the repressors for 5-HT1A in the serotonergic raphe neurons, and are also altered in postmortem brains of individuals with major depressive disorder (MDD) and in rats exposed to chronic restraint stress. We sought to find out if rats exposed to chronic social defeat (CSD) stress also show altered expression of these genes. Adult male Wistar rats were exposed to CSD stress for four consecutive weeks following which they were sacrificed and gene expression assessed in the prefrontal cortex (PFC) by quantitative real-time polymerase chain reaction. While CSD had no significant effects on NUDR and Freud-1 mRNA levels, 5-HT1A mRNA levels were significantly downregulated in defeated animals. The data suggest that regulatory factors other than Freud-1 and NUDR may be involved in the regulation of 5-HT1A expression in PFC during CSD stress. Furthermore, decreased levels of 5-HT1A following social defeat in the PFC are consistent with human postmortem results for this receptor in major depression and demonstrate the possibility that this receptor is involved in the pathophysiology of depression and other stress related disorders.

Elevated level of metabotropic glutamate receptor 2/3 in the prefrontal cortex in major depression.

Clinical, postmortem and preclinical research strongly implicates dysregulation of glutamatergic neurotransmission in major depressive disorder (MDD). Recently, metabotropic glutamate receptors (mGluRs) have been proposed as attractive targets for the discovery of novel therapeutic approaches against depression. The aim of this study was to examine mGluR2/3 protein levels in the prefrontal cortex (PFC) from depressed subjects. In addition, to test whether antidepressants influence mGluR2/3 expression we also studied levels of mGluR2/3 in fluoxetine-treated monkeys. Postmortem human prefrontal samples containing Brodmann's area 10 (BA10) were obtained from 11 depressed and 11 psychiatrically healthy controls. Male rhesus monkeys were treated chronically with fluoxetine (dose escalated to 3mg/kg, p.o.; n=7) or placebo (n=6) for 39 weeks. The mGluR2/3 immunoreactivity was investigated using Western blot method. There was a robust (+67%) increase in the expression of the mGlu2/3 protein in the PFC of depressed subjects relative to healthy controls. The expression of mGlu2/3 was unchanged in the PFC of monkeys treated with fluoxetine. Our findings provide the first evidence that mGluR2/3 is elevated in the PFC in MDD. This observation is consistent with reports showing that mGluR2/3 antagonists exhibit antidepressant-like activity in animal models and demonstrates that these receptors are promising targets for the discovery of novel antidepressants.

Expression of arginine decarboxylase in brain regions and neuronal cells.

After our initial report of a mammalian gene for arginine decarboxylase, an enzyme for the synthesis of agmatine from arginine, we have determined the regional expression of ADC in rat. We have analyzed the expression of ADC in rat brain regions by activity, protein and mRNA levels, and the regulation of expression in neuronal cells by RNA interference. In rat brain, ADC was widely expressed in major brain regions, with a substantial amount in hypothalamus, followed by cortex, and with least amounts in locus coeruleus and medulla. ADC mRNA was detected in primary astrocytes and C6 glioma cells. While no ADC message was detected in fresh neurons (3 days old), significant message appeared in differentiated neurons (3 weeks old). PC12 cells, treated with nerve growth factor, had higher ADC mRNA compared with naive cells. The siRNA mixture directed towards the N-terminal regions of ADC cDNA down-regulated the levels of mRNA and protein in cultured neurons/C6 glioma cells and these cells produced lower agmatine. Thus, this study demonstrates that ADC message is expressed in rat brain regions, that it is regulated in neuronal cells and that the down-regulation of ADC activity by specific siRNA leads to lower agmatine production.

Age-associated changes in mRNA levels of Phox2, norepinephrine transporter and dopamine beta-hydroxylase in the locus coeruleus and adrenal glands of rats.

Age-related changes in the gene expression of the transcription factors, Phox2a and 2b, and two marker proteins, norepinephrine transporter (NET) and dopamine beta-hydroxylase (DBH), of noradrenergic neurons were characterized in the locus coeruleus (LC) and adrenal glands using in situ hybridization. Analysis of changes was performed in rats that were 1-23 months of age. Compared to 1-month-old rats, there was a 62% increase of Phox2a messenger RNA (mRNA) in the LC of 3-month-old rats, and a decline of 37% in 23-month-old rats. In contrast, levels of Phox2b mRNA in the LC remained unchanged in 3-month-old rats, but declined to a 30% reduction in 23-month-old rats. Interestingly, mRNA levels of NET in the LC decreased with increasing age to a reduction of 29%, 30% and 43% in 3-, 8- and 23-month-old rats, respectively. Similarly, DBH mRNA in the LC declined with increasing age to a 56% reduction in 23-month-old rats. mRNA levels of Phox2a, Phox2b, NET and DBH in the adrenal medulla of 23-month-old rats were significantly lower than those of 1-month-old rats. Semi-quantitative reverse transcription assays of the same genes yielded data similar to in situ hybridization experiments, with beta-actin mRNA levels being unchanged across the ages. Taken together, these data reveal that reduced Phox2 mRNAs in the LC and adrenal medulla of aging rats are accompanied by a coincidental decline in mRNA levels of NET and DBH and suggest a possible relationship between Phox2 genes and the marker genes in noradrenergic neurons after birth.

Regional distribution and cellular localization of the ETS-domain transcription factor, FEV, mRNA in the human postmortem brain.

The ETS transcription factor Fifth Ewing Variant (FEV) mRNA, a homologue of the rodent Pet-1 gene that is exclusively expressed in serotonin-containing neurons and is a critical determinant of serotonin neuronal differentiation and development, was examined in human postmortem brain tissue using in situ hybridization histochemistry. Studies revealed that FEV mRNA is robustly and exclusively expressed in the major serotonin-containing cell groups of the dorsal and median raphe nuclei located in the midbrain and pons of the human brainstem. The localization of transcription factor, FEV, mRNA in serotonin-containing neurons of the human brain raises questions regarding the functional significance of this transcription factor in regulating serotonin-related genes and its potential role in psychiatric illness.