Key role of the 5-HT1A receptor addressing protein Yif1B in serotonin neurotransmission and SSRI treatment.

Background: Altered function of serotonin receptor 1A (5-HT1AR) has been consistently implicated in anxiety, major depressive disorder and resistance to antidepressants. Mechanisms by which the function of 5-HT1AR (expressed as an autoreceptor in serotonergic raphe neurons and as a heteroreceptor in serotonin [5-HT] projection areas) is altered include regulation of its expression, but 5-HT1AR trafficking may also be involved. Methods: We investigated the consequences of the lack of Yif1B (the 5-HT1AR trafficking protein) on 5-HT neurotransmission in mice, and whether Yif1B expression might be affected under conditions known to alter 5-HT neurotransmission, such as anxious or depressive states or following treatment with fluoxetine (a selective serotonin reuptake inhibitor) in humans, monkeys and mice. Results: Compared with wild-type mice, Yif1B-knockout mice showed a significant decrease in the forebrain density of 5-HT projection fibres and a hypofunctionality of 5-HT1A autoreceptors expressed on raphe 5-HT neurons. In addition, social interaction was less in Yif1B-knockout mice, which did not respond to the antidepressant-like effect of acute fluoxetine injection. In wild-type mice, social defeat was associated with downregulated Yif1B mRNA in the prefrontal cortex, and chronic fluoxetine treatment increased Yif1B expression. The expression of Yif1B was also downregulated in the postmortem prefrontal cortex of people with major depressive disorder and upregulated after chronic treatment with a selective serotonin reuptake inhibitor in monkeys. Limitations: We found sex differences in Yif1B expression in humans and monkeys, but not in mice under the tested conditions. Conclusion: These data support the concept that Yif1B plays a critical role in 5-HT1AR functioning and brain 5-HT homeostasis. The opposite changes in its expression observed in anxious or depressive states and after therapeutic fluoxetine treatment suggest that Yif1B might be involved in vulnerability to anxiety and depression, and fluoxetine efficacy.

Mechanistic role for a novel glucocorticoid-KLF11 (TIEG2) protein pathway in stress-induced monoamine oxidase A expression.

Chronic stress is a risk factor for psychiatric illnesses, including depressive disorders, and is characterized by increased blood glucocorticoids and brain monoamine oxidase A (MAO A, which degrades monoamine neurotransmitters). This study elucidates the relationship between stress-induced MAO A and the transcription factor Kruppel-like factor 11 (KLF11, also called TIEG2, a member of the Sp/KLF- family), which inhibits cell growth. We report that 1) a glucocorticoid (dexamethasone) increases KLF11 mRNA and protein levels in cultured neuronal cells; 2) overexpressing KLF11 increases levels of MAO A mRNA and enzymatic activity, which is further enhanced by glucocorticoids; in contrast, siRNA-mediated KLF11 knockdown reduces glucocorticoid-induced MAO A expression in cultured neurons; 3) induction of KLF11 and translocation of KLF11 from the cytoplasm to the nucleus are key regulatory mechanisms leading to increased MAO A catalytic activity and mRNA levels because of direct activation of the MAO A promoter via Sp/KLF-binding sites; 4) KLF11 knockout mice show reduced MAO A mRNA and catalytic activity in the brain cortex compared with wild-type mice; and 5) exposure to chronic social defeat stress induces blood glucocorticoids and activates the KLF11 pathway in the rat brain, which results in increased MAO A mRNA and enzymatic activity. Thus, this study reveals for the first time that KLF11 is an MAO A regulator and is produced in response to neuronal stress, which transcriptionally activates MAO A. The novel glucocorticoid-KLF11-MAO A pathway may play a crucial role in modulating distinct pathophysiological steps in stress-related disorders.

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.

Rational and Translational Implications of D-Amino Acids for Treatment-Resistant Schizophrenia: From Neurobiology to the Clinics.

Schizophrenia has been conceptualized as a neurodevelopmental disorder with synaptic alterations and aberrant cortical-subcortical connections. Antipsychotics are the mainstay of schizophrenia treatment and nearly all share the common feature of dopamine D2 receptor occupancy, whereas glutamatergic abnormalities are not targeted by the presently available therapies. D-amino acids, acting as N-methyl-D-aspartate receptor (NMDAR) modulators, have emerged in the last few years as a potential augmentation strategy in those cases of schizophrenia that do not respond well to antipsychotics, a condition defined as treatment-resistant schizophrenia (TRS), affecting almost 30-40% of patients, and characterized by serious cognitive deficits and functional impairment. In the present systematic review, we address with a direct and reverse translational perspective the efficacy of D-amino acids, including D-serine, D-aspartate, and D-alanine, in poor responders. The impact of these molecules on the synaptic architecture is also considered in the light of dendritic spine changes reported in schizophrenia and antipsychotics' effect on postsynaptic density proteins. Moreover, we describe compounds targeting D-amino acid oxidase and D-aspartate oxidase enzymes. Finally, other drugs acting at NMDAR and proxy of D-amino acids function, such as D-cycloserine, sarcosine, and glycine, are considered in the light of the clinical burden of TRS, together with other emerging molecules.

Linking Inflammation, Aberrant Glutamate-Dopamine Interaction, and Post-synaptic Changes: Translational Relevance for Schizophrenia and Antipsychotic Treatment: a Systematic Review.

Evidence from clinical, preclinical, and post-mortem studies supports the inflammatory/immune hypothesis of schizophrenia pathogenesis. Less evident is the link between the inflammatory background and two well-recognized functional and structural findings of schizophrenia pathophysiology: the dopamine-glutamate aberrant interaction and the alteration of dendritic spines architecture, both believed to be the "quantal" elements of cortical-subcortical dysfunctional network. In this systematic review, we tried to capture the major findings linking inflammation, aberrant glutamate-dopamine interaction, and post-synaptic changes under a direct and inverse translational perspective, a paramount picture that at present is lacking. The inflammatory effects on dopaminergic function appear to be bidirectional: the inflammation influences dopamine release, and dopamine acts as a regulator of discrete inflammatory processes involved in schizophrenia such as dysregulated interleukin and kynurenine pathways. Furthermore, the link between inflammation and glutamate is strongly supported by clinical studies aimed at exploring overactive microglia in schizophrenia patients and maternal immune activation models, indicating impaired glutamate regulation and reduced N-methyl-D-aspartate receptor (NMDAR) function. In addition, an inflammatory/immune-induced alteration of post-synaptic density scaffold proteins, crucial for downstream NMDAR signaling and synaptic efficacy, has been demonstrated. According to these findings, a significant increase in plasma inflammatory markers has been found in schizophrenia patients compared to healthy controls, associated with reduced cortical integrity and functional connectivity, relevant to the cognitive deficit of schizophrenia. Finally, the link between altered inflammatory/immune responses raises relevant questions regarding potential new therapeutic strategies specifically for those forms of schizophrenia that are resistant to canonical antipsychotics or unresponsive to clozapine.

Transcriptional expression of serotonergic regulators in laser-captured microdissected dorsal raphe neurons of subjects with major depressive disorder: sex-specific differences.

The relationship between serotonin (5-HT) and major depressive disorder (MDD) has been extensively studied but certain aspects are still ambiguous. Given the evidence that 5-HT neurotransmission is reduced in depressed subjects, it is possible that one or more of the 5-HT regulators may be altered in the dorsal raphe nucleus (DR) of depressed subjects. Candidates that regulate 5-HT synthesis and neuronal activity of 5-HT neurons include intrinsic regulators such as tryptophan hydroxylase 2, 5-HT autoreceptors, 5-HT transporter and transcription factors, as well as afferent regulators such as estrogen and brain-derived neurotrophic factor. The present study was designed to quantify mRNA concentrations of the above 5-HT regulators in an isolated population of 5-HT-containing DR neurons of MDD subjects and gender-matched psychiatrically normal control subjects. We found that mRNA concentrations of the 5-HT1D receptor and the transcription factors, NUDR and REST, were significantly increased in DR-captured neurons of female MDD subjects compared to female control subjects. No significant differences were found for the transcripts in male MDD subjects compared to male controls. This study reveals sex-specific alterations in gene expression of the pre-synaptic 5-HT1D autoreceptors and 5-HT-related transcription factors, NUDR and REST, in DR neurons of women with MDD.

Decreased expression of Freud-1/CC2D1A, a transcriptional repressor of the 5-HT1A receptor, in the prefrontal cortex of subjects with major depression.

Serotonin1A (5-HT(1A)) receptors are reported altered in the brain of subjects with major depressive disorder (MDD). Recent studies have identified transcriptional regulators of the 5-HT(1A) receptor and have documented gender-specific alterations in 5-HT(1A) transcription factor and 5-HT(1A) receptors in female MDD subjects. The 5' repressor element under dual repression binding protein-1 (Freud-1) is a calcium-regulated repressor that negatively regulates the 5-HT(1A) receptor gene. This study documented the cellular expression of Freud-1 in the human prefrontal cortex (PFC) and quantified Freud-1 protein in the PFC of MDD and control subjects as well as in the PFC of rhesus monkeys chronically treated with fluoxetine. Freud-1 immunoreactivity was present in neurons and glia and was co-localized with 5-HT(1A) receptors. Freud-1 protein level was significantly decreased in the PFC of male MDD subjects (37%, p=0.02) relative to gender-matched control subjects. Freud-1 protein was also reduced in the PFC of female MDD subjects (36%, p=0.18) but was not statistically significant. When the data was combined across genders and analysed by age, the decrease in Freud-1 protein level was greater in the younger MDD subjects (48%, p=0.01) relative to age-matched controls as opposed to older depressed subjects. Similarly, 5-HT(1A) receptor protein was significantly reduced in the PFC of the younger MDD subjects (48%, p=0.01) relative to age-matched controls. Adult male rhesus monkeys administered fluoxetine daily for 39 wk revealed no significant change in cortical Freud-1 or 5-HT(1A) receptor proteins compared to vehicle-treated control monkeys. Reduced protein expression of Freud-1 in MDD subjects may reflect dysregulation of this transcription factor, which may contribute to the altered regulation of 5-HT(1A) receptors observed in subjects with MDD. These data may also suggest that reductions in Freud-1 protein expression in the PFC may be associated with early onset of MDD.

Gender-specific decrease in NUDR and 5-HT1A receptor proteins in the prefrontal cortex of subjects with major depressive disorder.

A variety of studies have documented alterations in 5-HT1A receptor binding sites in the brain of subjects with major depressive disorder (MDD). The recently identified transcription factor, nuclear deformed epidermal autoregulatory factor (NUDR/Deaf-1) has been shown to function as a transcriptional modulator of the human 5-HT1A receptor gene. The present study was undertaken to document the regional and cellular localization of NUDR in the human prefrontal cortex and to examine the levels of NUDR and 5-HT1A receptor protein in prefrontal cortex of female and male depressed and control subjects. NUDR immunoreactivity was present in neurons and glia across cortical layers and was co-localized with 5-HT1A receptor immunoreactive neurons. NUDR immunoreactivity as measured by Western blot was significantly decreased in the prefrontal cortex of female depressed subjects (42%, p=0.02) and unchanged in male depressed subjects relative to gender-matched control subjects. Similarly, 5-HT1A receptor protein level was significantly reduced in the prefrontal cortex of female depressed subjects (46%, p=0.03) and unchanged in male depressed subjects compared to gender-matched control subjects. Reduced protein expression of NUDR in the prefrontal cortex of female subjects with MDD may reflect a functional alteration in this transcription factor, which may contribute to the decrease in 5-HT1A receptors observed in the same female subjects with MDD. In addition, the gender-specific alterations in cortical NUDR and 5-HT1A receptor proteins could represent an underlying biological mechanism associated with the higher incidence of depression in women.

Length of axons expressing the serotonin transporter in orbitofrontal cortex is lower with age in depression.

Studies of major depressive disorder (MDD) in postmortem brain tissue report enhanced binding to inhibitory serotonin-1A autoreceptors in midbrain dorsal raphe and reductions in length of axons expressing the serotonin transporter (SERT) in dorsolateral prefrontal cortex. The length density of axons expressing SERT in the orbitofrontal cortex (OFC) was determined in 18 subjects with MDD and 17 age-matched control subjects. A monoclonal antibody was used to immunohistochemically label the SERT in fixed sections of OFC. The 3-dimensional length density of SERT-immunoreactive (ir) axons in layer VI of OFC was estimated. The age of subjects with MDD was negatively correlated with SERT axon length (r=-0.77, p<0.0005). The significant effect of age persisted when removing four depressed subjects with an antidepressant medication present at the time of death, or when removing nine depressed subjects that had a recent prescription for an antidepressant medication. Neither gender, tissue pH, postmortem interval, 5-HTTLPR genotype, time in fixative, nor death by suicide had a significant effect on axon length. The age-related decrease in SERT-ir axon length in MDD may reflect pathology of ascending axons passing through deep white matter hyperintensities. Greater length of axons expressing SERT in younger subjects with MDD may result in a significant deficit in serotonin availability in OFC.

Effect of 6-hydroxydopamine on striatal GDNF and nigral GFRalpha1 and RET mRNAs in the adult rat.

Exogenous GDNF as well as vectors containing the gene for this trophic factor has been shown to be neuroprotective in animal models of Parkinson's disease. We therefore investigated whether changes in striatal GDNF protein and nigral mRNA levels of its co-receptors GFRalpha1 and RET occur in response to lesions of dopamine (DA) neurons and examined the temporal profile of these changes as they relate to the loss of dopaminergic markers. Rats were lesioned with 6-hydroxydopamine and sacrificed 3 h to 60 days post-infusion. DA tissue levels in the striatum and tyrosine hydroxylase immunoreactivity in the substantia nigra (SN) and ventral tegmental area (VTA) were used to determine the size of the lesions. GDNF protein was measured in the striatum using radioimmunocytochemistry. In situ hybridization was used to determine alterations in the mRNAs of RET and GFRalpha1 in the SN and VTA. We observed no persistent changes in GDNF protein in the striatum in response to 6-hydroxydopamine over the 60-day observation period, suggesting that compensatory changes in this trophic factor do not occur in response to injury. Dramatic decreases in RET and GFRalpha1 were observed in both SN and VTA that were generally correlated with the loss of TH protein and striatal DA content, strongly suggesting that these receptors are located on DA neurons and that the protective effect of GDNF reflects a direct action of the trophic factor on these neurons.

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.

The reduction of R1, a novel repressor protein for monoamine oxidase A, in major depressive disorder.

The novel transcriptional repressor protein, R1 (JPO2/CDCA7L/RAM2), inhibits monoamine oxidase A (MAO A) gene expression and influences cell proliferation and survival. MAO A is implicated in several neuropsychiatric illnesses and highly elevated in major depressive disorder (MDD); however, whether R1 is involved in these disorders is unknown. This study evaluates the role of R1 in depressed subjects either untreated or treated with antidepressant drugs. R1 protein levels were determined in the postmortem prefrontal cortex of 18 untreated MDD subjects and 12 medicated MDD subjects compared with 18 matched psychiatrically normal control subjects. Western blot analysis showed that R1 was significantly decreased by 37.5% (p<0.005) in untreated MDD subjects. The R1 level in medicated MDD subjects was also significantly lower (by 30%; p<0.05) compared with control subjects, but was not significantly different compared with untreated MDD subjects. Interestingly, the reduction in R1 was significantly correlated with an increase (approximately 40%; p<0.05) in MAO A protein levels within the MDD groups compared with controls. Consistent with the change in MAO A protein expression, the MAO A catalytic activity was significantly greater in both MDD groups compared with controls. These results suggest that reduced R1 may lead to elevated MAO A levels in untreated and treated MDD subjects; moreover, the reduction of R1 has been implicated in apoptotic cell death and apoptosis has also been observed in the brains of MDD subjects. Therefore, modulation of R1 levels may provide a new therapeutic target in the development of more effective strategies to treat MDD.

Glial and glutamatergic markers in depression, alcoholism, and their comorbidity.

BACKGROUND: Alteration of glutamatergic neurotransmission in the prefrontal cortex (PFC) may contribute to the pathophysiology of alcoholism and major depressive disorder (MDD). Among glial cells, astrocytes are mostly responsible for recycling synaptic glutamate by uptake through excitatory amino acid transporters 1 and 2 (EAAT1 and EAAT2), and conversion to glutamine with glutamine synthetase (GS). Low density of astrocytes in the PFC of "uncomplicated' alcoholics and MDD subjects may parallel altered glutamate transporters and GS in the PFC. METHODS: Immunohistochemistry and Western blotting for glutamate transporters, GS and glial fibrillary acidic protein (GFAP) were applied to postmortem tissue of the left orbitofrontal cortex from 13 subjects with MDD, 13 with alcoholism, 10 with comorbid alcoholism plus MDD (MDA), and 13 non-psychiatric controls. Area fraction of immunoreactivity was measured in sections, and protein levels in Western blots. RESULTS: EAAT2 immunoreactivity was significantly lower in MDD and MDA subjects than in controls. EAAT1 levels were lower in MDA and MDD subjects as compared to controls, while GS levels in MDA were significantly lower than in alcoholics and controls, and lower in MDD subjects than in alcoholics. Area fraction of GFAP was lower in MDD, but not in MDA subjects as compared to controls or alcoholics. LIMITATIONS: High variability of protein levels in some groups and effects of antidepressant treatment, although appearing to be limited, cannot be fully evaluated. CONCLUSIONS: There are differential changes in the expression of glial glutamatergic markers in depression and alcoholism, suggesting a depletion of certain aspects of glutamatergic processing in depression.

A novel role for glyceraldehyde-3-phosphate dehydrogenase and monoamine oxidase B cascade in ethanol-induced cellular damage.

BACKGROUND: Alcoholism is a major psychiatric condition at least partly associated with ethanol (EtOH)-induced cell damage. Although brain cell loss has been reported in subjects with alcoholism, the molecular mechanism is unclear. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and monoamine oxidase B (MAO B) reportedly play a role in cellular dysfunction under stressful conditions and might contribute to EtOH-induced cell damage. METHODS: Expression of GAPDH and MAO B protein was studied in human glioblastoma and neuroblastoma cell lines exposed to physiological concentrations of EtOH. Expression of these proteins was also examined in the prefrontal cortex from human subjects with alcohol dependence and in rats fed with an EtOH diet. Coimmunoprecipitation, subcellular fractionation, and luciferase assay were used to address nuclear GAPDH-mediated MAO B activation. To test the effects of inactivation, RNA interference and pharmacological intervention were used, and cell damage was assessed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP Nick End Labeling (TUNEL) and hydrogen peroxide measurements. RESULTS: Ethanol significantly increases levels of GAPDH, especially nuclear GAPDH, and MAO B in neuronal cells as well as in human and rat brains. Nuclear GAPDH interacts with the transcriptional activator, transforming growth factor-beta-inducible early gene 2 (TIEG2), and augments TIEG2-mediated MAO B transactivation, which results in cell damage in neuronal cells exposed to EtOH. Knockdown expression of GAPDH or treatment with MAO B inhibitors selegiline (deprenyl) and rasagiline (Azilect) can block this cascade. CONCLUSIONS: Ethanol-elicited nuclear GAPDH augments TIEG2-mediated MAO B, which might play a role in brain damage in subjects with alcoholism. Compounds that block this cascade are potential candidates for therapeutic strategies.

Human Freud-2/CC2D1B: a novel repressor of postsynaptic serotonin-1A receptor expression.

BACKGROUND: Altered expression of serotonin-1A (5-HT1A) receptors, both presynaptic in the raphe nuclei and post-synaptic in limbic and cortical target areas, has been implicated in mood disorders such as major depression and anxiety. Within the 5-HT1A receptor gene, a powerful dual repressor element (DRE) is regulated by two protein complexes: Freud-1/CC2D1A and a second, unknown repressor. Here we identify human Freud-2/CC2D1B, a Freud-1 homologue, as the second repressor. METHODS: Freud-2 distribution was examined with Northern and Western blot, reverse transcriptase polymerase chain reaction, and immunohistochemistry/immunofluorescence; Freud-2 function was examined by electrophoretic mobility shift, reporter assay, and Western blot. RESULTS: Freud-2 RNA was widely distributed in brain and peripheral tissues. Freud-2 protein was enriched in the nuclear fraction of human prefrontal cortex and hippocampus but was weakly expressed in the dorsal raphe nucleus. Freud-2 immunostaining was co-localized with 5-HT1A receptors, neuronal and glial markers. In prefrontal cortex, Freud-2 was expressed at similar levels in control and depressed male subjects. Recombinant hFreud-2 protein bound specifically to 5' or 3' human DRE adjacent to the Freud-1 site. Human Freud-2 showed strong repressor activity at the human 5-HT1A or heterologous promoter in human HEK-293 5-HT1A-negative cells and neuronal SK-N-SH cells, a model of postsynaptic 5-HT1A receptor-positive cells. Furthermore, small interfering RNA knockdown of endogenous hFreud-2 expression de-repressed 5-HT1A promoter activity and increased levels of 5-HT1A receptor protein in SK-N-SH cells. CONCLUSIONS: Human Freud-2 binds to the 5-HT1A DRE and represses the human 5-HT1A receptor gene to regulate its expression in non-serotonergic cells and neurons.

Increased tryptophan hydroxylase immunoreactivity in the dorsal raphe nucleus of alcohol-dependent, depressed suicide subjects is restricted to the dorsal subnucleus.

Considerable evidence suggests that alcoholics with co-occurring depressive disorder are at greater risk for developing psychosocial problems particularly suicidal behavior. Moreover, dysfunction in serotonin (5-HT) neurotransmission has been implicated in depression, suicide and alcoholism. In the present study, we measured the levels of tryptophan hydroxylase (TPH), the main synthetic enzyme of 5-HT synthesis, in specific nuclei of the dorsal raphe (DR) in depressed suicide victims with alcohol dependence and matched psychiatrically normal controls. TPH immunoreactivity (IR) was quantified in frozen tissue sections containing the DR from 8 suicide victims with a diagnosis of major depression and alcohol dependence, and 8 psychiatrically normal control subjects by using immunoautoradiographic methods. We found that the levels of TPH-IR were significantly increased by 46% in the dorsal subnucleus of the DR in depressed suicide victims with alcohol dependence when compared with controls. In contrast, TPH-IR levels did not significantly differ in the other DR subnuclei between depressed, alcoholic suicide subjects, and controls. Our results indicate that abnormalities in 5-HT biosynthesis in the brain of depressed alcoholic suicide subjects are restricted within distinct regions of the DR.

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.