Spinophilin expression in postmortem prefrontal cortex of schizophrenic subjects: Effects of antipsychotic treatment.

Schizophrenia has been associated with alterations in neurotransmission and synaptic dysfunction. Spinophilin is a multifunctional scaffold protein that modulates excitatory synaptic transmission and dendritic spine morphology. Spinophilin can also directly interact with and regulate several receptors for neurotransmitters, such as dopamine D2 receptors, which play a role in the pathophysiology of schizophrenia and are targets of antipsychotics. Several studies have thus suggested an implication of spinophilin in schizophrenia. In the present study spinophilin protein expression was determined by western blot in the postmortem dorsolateral prefrontal cortex of 24 subjects with schizophrenia (12 antipsychotic-free and 12 antipsychotic-treated subjects) and 24 matched controls. Experiments were performed in synaptosomal membranes (SPM) and in postsynaptic density fractions (PSD). As previously reported, two specific bands for this protein were observed: an upper 120-130 kDa band and a lower 80-95 kDa band. The spinophilin lower band showed a significant decrease in schizophrenia subjects compared to matched controls, both in SPM and PSD fractions (-15%, p = 0.007 and -15%, p = 0.039, respectively). When schizophrenia subjects were divided by the presence or absence of antipsychotics in blood at death, the lower band showed a significant decrease in antipsychotic-treated schizophrenia subjects (-24%, p = 0.003 for SPM and -26%, p = 0.014 for PSD), but not in antipsychotic-free subjects, compared to their matched controls. These results suggest that antipsychotics could produce alterations in spinophilin expression that do not seem to be related to schizophrenia per se. These changes may underlie some of the side effects of antipsychotics.

Functional approaches to the study of G-protein-coupled receptors in postmortem brain tissue: [35S]GTPγS binding assays combined with immunoprecipitation.

G-protein-coupled receptors (GPCRs) have an enormous biochemical importance as they bind to diverse extracellular ligands and regulate a variety of physiological and pathological responses. G-protein activation measures the functional consequence of receptor occupancy at one of the earliest receptor-mediated events. Receptor coupling to G-proteins promotes the GDP/GTP exchange on Gα subunits. Thus, modulation of the binding of the poorly hydrolysable GTP analog [35S]GTPγS to the Gα-protein subunit can be used as a functional approach to quantify GPCR interaction with agonist, antagonist or inverse agonist drugs. In order to determine receptor-mediated selective activation of the different Gα-proteins, [35S]GTPγS binding assays combined with immunodetection by specific antibodies have been developed and applied to physiological and pathological brain conditions. Currently, immunoprecipitation with magnetic beads and scintillation proximity assays are the most habitual techniques for this purpose. The present review summarizes the different procedures, advantages and limitations of the [35S]GTPγS binding assays combined with selective Gα-protein sequestration methods. Experience of functional coupling of several GPCRs to different Gα-proteins and recommendations for optimal performance in brain membranes are described. One of the biggest opportunities opened by these techniques is that they enable evaluation of biased agonism in the native tissue, which results in high interest in drug discovery. The available results derived from application of these functional methodologies to study GPCR dysfunctions in neuro-psychiatric disorders are also described. In conclusion, [35S]GTPγS binding combined with antibody-mediated immunodetection represents an useful method to separately evaluate the functional activity of drugs acting on GPCRs over each Gα-protein subtype.

α2A- and α2C-adrenoceptor expression and functionality in postmortem prefrontal cortex of schizophrenia subjects.

Previous evidence suggests that α2-adrenoceptors (α2-AR) may be involved in the pathophysiology of schizophrenia. However, postmortem brain studies on α2-AR expression and functionality in schizophrenia are scarce. The aim of our work was to evaluate α2A-AR and α2C-AR expression in different subcellular fractions of prefrontal cortex postmortem tissue from antipsychotic-free (absence of antipsychotics in blood at the time of death) (n = 12) and antipsychotic-treated (n = 12) subjects with schizophrenia, and matched controls (n = 24). Functional coupling of α2-AR to Gα proteins induced by the agonist UK14304 was also tested. Additionally, Gα protein expression was also evaluated. In antipsychotic-free schizophrenia subjects, α2A-AR and α2C-AR protein expression was similar to controls in all the subcellular fractions. Conversely, in antipsychotic-treated schizophrenia subjects, increased α2A-AR expression was found in synaptosomal plasma membrane and postsynaptic density (PSD) fractions (+60% and +79% vs controls, respectively) with no significant changes in α2C-AR. [35S]GTPγS SPA experiments showed a significant lower stimulation of Gαi2 and Gαi3 proteins by UK14304 in antipsychotic-treated schizophrenia subjects, whereas stimulation in antipsychotic-free schizophrenia subjects remained unchanged. Gαo protein stimulation was significantly decreased in both antipsychotic-free and antipsychotic-treated schizophrenia subjects compared to controls. Expression of Gαi3 protein did not differ between groups, whereas Gαi2 levels were increased in PSD of schizophrenia subjects, both antipsychotic-free and antipsychotic-treated. Gαo protein expression was increased in PSD of antipsychotic-treated subjects and in the presynaptic fraction of antipsychotic-free schizophrenia subjects. The present results suggest that antipsychotic treatment is able to modify in opposite directions both the protein expression and the functionality of α2A-AR in the cortex of schizophrenia patients.

Differential α2A- and α2C-adrenoceptor protein expression in presynaptic and postsynaptic density fractions of postmortem human prefrontal cortex.

BACKGROUND: Three different α2-adrenoceptor (α2-AR) subtypes have been described. The α2A-AR and α2C-AR subtypes are highly expressed in the human prefrontal cortex, where they modulate neurotransmission. However, due to the lack of subtype-selective ligands, the physiological relevance of both subtypes has not been fully resolved. AIMS: In this context, the aim of the present study was to characterize the protein expression of both α2-AR subtypes, in different synaptic fractions of postmortem human prefrontal cortex. METHODS: A subcellular fractionation of the samples was performed and the protein expression of α2A- and α2C-ARs was measured in presynaptic membranes and postsynaptic density fractions by Western blot. RESULTS: The results revealed that the α2A-AR subtype is mainly located postsynaptically (95±3%) whereas the remaining 5±3% is in the presynapse. Conversely, the α2C-AR subtype showed a similar distribution between pre- and postsynaptic membranes, with a slightly higher percentage present in the presynapse (60±2% vs. 40±2%). CONCLUSIONS: These findings could explain some contradictory effects reported for α2-AR agonists and antagonists in the human prefrontal cortex. Furthermore, the present data could contribute to elucidating the therapeutic potential of selectively targeting α2A- or α2C-AR subtypes.

Serotonin 5-HT2A receptor expression and functionality in postmortem frontal cortex of subjects with schizophrenia: Selective biased agonism via Gαi1-proteins.

Serotonin 5-HT2A receptors (5-HT2ARs) have been implicated in schizophrenia. However, postmortem studies on 5-HT2ARs expression and functionality in schizophrenia are scarce. The 5-HT2AR mRNA and immunoreactive protein expression were evaluated in postmortem tissue from dorsolateral prefrontal cortex (DLPFC) of antipsychotic-free (n = 18) and antipsychotic-treated (n = 9) subjects with schizophrenia, and matched controls (n = 27). Functional coupling of 5-HT2AR to G-proteins was tested by measuring the activation induced by the agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride ((±)DOI) in antibody-capture [35S]GTPγS scintillation proximity assays (SPA). In antipsychotic-free schizophrenia subjects, 5-HT2AR mRNA expression and protein immunoreactivity in total homogenates was similar to controls. In contrast, in antipsychotic-treated schizophrenia subjects, lower mRNA expression (60±9% vs controls) and a trend to reduced protein immunoreactivity (86±5% vs antipsychotic-free subjects) just in membrane-enriched fractions was observed. [35S]GTPγS SPA revealed a significant ~6% higher stimulation of Gαi1-protein by (±)DOI in schizophrenia, whereas activation of the canonical Gαq/11-protein pathway by (±)DOI remained unchanged. Expression of Gαi1- and Gαq/11-proteins did not differ between groups. Accordingly, in rats chronically treated with clozapine, but not with haloperidol, a 30-40% reduction was observed in 5-HT2AR mRNA expression, 5-HT2AR protein immunoreactivity and [3H]ketanserin binding in brain cortical membranes. Overall, the data suggest a supersensitive 5-HT2AR signaling through inhibitory Gαi1-proteins in schizophrenia. Together with previous results, a dysfunctional pro-hallucinogenic agonist-sensitive 5-HT2AR conformation in postmortem DLPFC of subjects with schizophrenia is proposed. Atypical antipsychotic treatment would contribute to counterbalance this 5-HT2AR supersensitivity by reducing receptor expression.

Monoamine oxidase B activity is increased in human gliomas.

Glial tumours are the most common type of brain neoplasm in humans. Tumour classification and grading represent key factors for patient management. However, current grading schemes are still limited by subjective histological criteria. In this context, gliosis has been linked to increases in monoamine oxidase B (MAO-B) activity. Thus, in the present study, MAO-B activity in membranes of glial tumours (n=20), meningiomas (n=12) and non-pathological human brains (n=15) was quantified by [14C]PEA oxidation. MAO-B activity was significantly greater in glioblastoma multiformes than in postmortem control brains (p<0.01) or meningiomas (p<0.001). There were no significant differences in MAO-B activity between glioblastoma multiformes (n=11) and low-grade astrocytomas (n=3) or anaplastic astrocytomas (n=6). In conclusion, the present results demonstrate a significant and selective increase in MAO-B activity in human gliomas when compared with meningiomas or non-tumoural tissue. These results suggest that the quantification of MAO-B activity may be a useful diagnostic tool for differentiating glial tumours from other types of brain tumours or surrounding normal brain tissue.

Alpha2C-adrenoceptor Del322-325 polymorphism and risk of psychiatric disorders: significant association with opiate abuse and dependence.

Objectives α2C-adrenoceptors (α2C-AR) are involved in behavioural responses relevant to psychiatric disorders and suicide completion. The genetic polymorphism α2CDel322-325-AR confers a loss-of-function phenotype. Functional human studies have associated α2CDel322-325-AR polymorphism with major depression pathophysiology. The aim of this study was to analyse, for the first time, the association of α2CDel322-325-AR polymorphism with suicide completion and with related psychiatric disorders: major depression, schizophrenia, opiate and alcohol abuse and dependence. Methods Post-mortem brain DNA was extracted (n = 516) and genotyping performed by HaeIII restriction endonuclease digestion of PCR products and DNA fragment analysis on capillary sequencer. Amplified products were sequenced to confirm the presence of the polymorphism. Results The frequency of α2CDel322-325-AR in suicide (9%, n = 236) and non-suicide victims (11%, n = 280) was similar. Genotype frequencies for the α2CDel322-325-AR polymorphism in depressed (15%, n = 39) and schizophrenic subjects (18%, n = 39) were higher than in controls (7%, n = 187), but these differences did not reach statistical significance (P = 0.125 and P = 0.063, respectively). A selective and significant association of α2CDel322-325-AR polymorphism with opiate abuse and dependence was found (23%, n = 35, P = 0.011). Conclusions Our results indicate that α2CDel322-325-AR may play a role in the pathophysiology of opiate abuse and dependence and raise the interest for larger genetic associative studies.

Up-regulated 14-3-3ß and 14-3-3ζ proteins in prefrontal cortex of subjects with schizophrenia: effect of psychotropic treatment.

14-3-3 is a family of conserved regulatory proteins that bind to a multitude of functionally diverse signalling proteins. Various genetic studies and gene expression and proteomic analyses have involved 14-3-3 proteins in schizophrenia (SZ). On the other hand, studies about the status of these proteins in major depressive disorder (MD) are still missing. Immunoreactivity values of cytosolic 14-3-3ß and 14-3-3ζ proteins were evaluated by Western blot in prefrontal cortex (PFC) of subjects with schizophrenia (SZ; n=22), subjects with major depressive disorder (MD; n=21) and age-, gender- and postmortem delay-matched control subjects (n=52). The modulation of 14-3-3ß and 14-3-3ζ proteins by psychotropic medication was also assessed. The analysis of both proteins in SZ subjects with respect to matched control subjects showed increased 14-3-3ß (Δ=33±10%, p<0.05) and 14-3-3ζ (Δ=29±6%, p<0.05) immunoreactivity in antipsychotic-free but not in antipsychotic-treated SZ subjects. Immunoreactivity values of 14-3-3ß and 14-3-3ζ were not altered in MD subjects. These results show the specific up-regulation of 14-3-3ß and 14-3-3ζ proteins in PFC of SZ subjects and suggest a possible down-regulation of both proteins by antipsychotic treatment.

Increased α2- and ß1-adrenoceptor densities in postmortem brain of subjects with depression: differential effect of antidepressant treatment.

BACKGROUND: Brain α2- and ß-adrenoceptor alterations have been suggested in suicide and major depressive disorder. METHODS: The densities of α2-, ß1- and ß2-adrenoceptors in postmortem prefrontal cortex of 26 subjects with depression were compared with those of age-, gender- and postmortem delay-matched controls. The effect of antidepressant treatment on α2- and ß-adrenoceptor densities was also evaluated. α2- and ß-adrenoceptor densities were measured by saturation experiments with respective radioligands [(3)H]UK14304 and [(3)H]CGP12177. ß1- and ß2-adrenoceptor subtype densities were dissected by means of ß1-adrenoceptor selective antagonist CGP20712A. RESULTS: Both, α2- and ß1-adrenoceptors densities were higher in antidepressant-free depressed subjects (n=14) than those in matched controls (Δ~24%, p=0.013 and Δ~20%, p=0.044, respectively). In antidepressant-treated subjects (n=12), α2-adrenoceptor density remained increased over that in controls (Δ~20%), suggesting a resistance of α2-adrenoceptors to the down-regulatory effect of antidepressants. By contrast, ß1-adrenoceptor density in antidepressant-treated depressed subjects was not different from controls, suggesting a possible down-regulation by antidepressants. The down-regulation of ß1-adrenoceptor density in antidepressant-treated depressed subjects differs from the unaltered ß1-adrenoceptor density observed in citalopram-treated rats and in a group of non-depressed subjects also treated with antidepressants (n=6). ß2-adrenoceptor density was not altered in depressed subjects independently of treatment. LIMITATIONS: Antidepressant-treated subjects had been treated with a heterogeneous variety of antidepressant drugs. The results should be understood in the context of suicide victims with depression. CONCLUSIONS: These results show the up-regulation of brain α2- and ß1-adrenoceptors in depression and suggest that the regulation induced by chronic antidepressant treatment would be altered in these subjects.

Differential regulation of RGS proteins in the prefrontal cortex of short- and long-term human opiate abusers.

Opiate addiction is characterized by drug tolerance and dependence which involve adaptive changes in µ-opioid receptors (MORs) signaling. Regulators of G-protein signaling RGS9, RGS4 and RGS10 proteins negatively regulate G(αi/o) protein activity modulating MOR function. An important role of RGS proteins in drug addiction has been described but the status of RGS proteins in human brain of opiate addicts remains unknown. The present study evaluated the immunoreactivity levels of RGS4, RGS9 and RGS10 proteins in prefrontal cortex of short- (n = 15) and long-term (n = 21) opiate abusers and in matched control subjects. RGS4 protein was not altered in short-term opiate abusers but, in long-term abusers it was significantly up-regulated (Δ = 29 ± 6%). RGS10 protein expression was significantly decreased in short-term (Δ = -42 ± 7%) but remained unaltered in long-term opiate abusers. RGS9 protein levels in opiate abusers did not differ from matched controls either in the short-term or in the long-term opiate abuser groups. RGS4, RGS9 and RGS10 levels were also studied in brains (frontal cortex) of rats submitted to acute and chronic morphine treatment and to spontaneous and naloxone-precipitated opiate withdrawal. Chronic morphine treatment in rats was associated with an increase in RGS4 protein immunoreactivity (Δ = 28 ± 7%), which persisted in spontaneous (Δ = 35 ± 8%) and naloxone-precipitated withdrawal (Δ = 30 ± 9%) without significant changes in RGS9 and RGS10 proteins. The specific modulation of RGS4 and RGS10 protein expression observed in the prefrontal cortex of opiate abusers might be relevant in the neurobiology of opiate tolerance, dependence and withdrawal. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.

HDAC2 regulates atypical antipsychotic responses through the modulation of mGlu2 promoter activity.

Histone deacetylases (HDACs) compact chromatin structure and repress gene transcription. In schizophrenia, clinical studies demonstrate that HDAC inhibitors are efficacious when given in combination with atypical antipsychotics. However, the molecular mechanism that integrates a better response to antipsychotics with changes in chromatin structure remains unknown. Here we found that chronic atypical antipsychotics downregulated the transcription of metabotropic glutamate 2 receptor (mGlu2, also known as Grm2), an effect that was associated with decreased histone acetylation at its promoter in mouse and human frontal cortex. This epigenetic change occurred in concert with a serotonin 5-HT(2A) receptor-dependent upregulation and increased binding of HDAC2 to the mGlu2 promoter. Virally mediated overexpression of HDAC2 in frontal cortex decreased mGlu2 transcription and its electrophysiological properties, thereby increasing psychosis-like behavior. Conversely, HDAC inhibitors prevented the repressive histone modifications induced at the mGlu2 promoter by atypical antipsychotics, and augmented their therapeutic-like effects. These observations support the view of HDAC2 as a promising new target for schizophrenia treatment.

Characterization of regulators of G-protein signaling RGS4 and RGS10 proteins in the postmortem human brain.

Regulator of G-protein signaling (RGS) proteins are a large family of proteins that accelerate GTPase rate of the Gα subunits and therefore, negatively regulate G-protein signaling. Expression of RGS4 and RGS10 proteins was characterized in human prefrontal cortex attending to methodological (subcellular localization, antibody specificity and sensitivity, postmortem delay (PMD) and storage conditions of the samples) and demographic issues (age and gender of the subjects). Anti-RGS4 (N-16) antibody revealed a unique and specific band of 38 kDa that was highly enriched in the plasma membrane. Anti-RGS10 (C-20) antibody revealed two specific bands of 24 and 27kDa, corresponding to two possible isoforms of this protein, which were predominantly localized in the cytosol. Antibody dilution and protein linearity studies confirmed the sensitivity of the signal. A large number of samples from 58 individuals presenting well spread PMD, storage time, age of the subjects at the time of death, and male and female distribution were studied. A positive linear relationship between the age and RGS4 immunoreactivity was observed. There was a negative influence of PMD on the RGS10 27 kDa band immunoreactivity but a positive relationship emerged between the PMD and RGS4 immunoreactivity. Storage time of the samples did not have any influence on RGS4 nor on RGS10 immunoreactivity, showing their stability at -70°C. When studying the RGS4 and RGS10 protein expression density in males and females, no significant difference was found between groups. This study demonstrates that RGS4 and RGS10 proteins can be detected by immunoreactive techniques in postmortem human brain cortex. The study provides important matching conditions that should be taken into account in postmortem brain studies of neuropsychiatric diseases.

Synthesis and pharmacological studies of new hybrid derivatives of fentanyl active at the mu-opioid receptor and I2-imidazoline binding sites.

Two series of fentanyl-derived hybrid molecules bearing potent I2-imidazoline binding site (IBS) ligands (i.e., guanidine and BU224 moieties) linked with an aliphatic (m=2, 3, 4, 6, 7, 8, 9 and 12 methylene units) or aromatic spacer were prepared. Their affinities for the mu-opioid receptors and for the I2-IBS were determined through competition binding studies on human postmortem brain membranes. Whereas the BU224 hybrid molecules bound to the mu-opioid receptor and the I2-IBS in the micromolar to low micromolar range, the alkaneguanidine series exhibited remarkable affinities in the nanomolar range for both receptors. [35S]GTPgammaS functional assays were performed on human postmortem brain membranes with selected ligands from each series (4f and 8g) showing the highest dual affinity for the mu-opioid receptor and I2-IBS affinities. Both compounds displayed agonist properties: at the mu-opioid receptor for the alkaneguanidine derivative 4f (spacer: six methylene units) and at a G-protein coupled receptor (GPCR) which remains to be determined for 8g. The lack of analgesic properties of 4f in vivo (i.e., hot plate and writhing tests in mice), discordant with the good in vitro binding data (Ki mu=1.04+/-0.28 nM, Ki I2=409+/-238 nM), may possibly be due to the low intrinsic efficacy of the compound. Alternatively, a low access to the central nervous system for this kind of hybrid molecules cannot be ruled out. Two new compounds reported here (9f and 13), which were not dual acting, are worth mentioning for their outstanding binding affinities; 9f bound to the mu-opioid receptor with a picomolar affinity (Ki=0.0098+/-0.0033 nM), whereas 13 presented an I2-IBS affinity (Ki=18+/-11 nM) similar to the reference compound BU224.