In need of a specific antibody against the oxytocin receptor for neuropsychiatric research: A KO validation study.

Antibodies are one of the most utilized tools in biomedical research. However, few of them are rigorously evaluated, as there are no accepted guidelines or standardized methods for determining their validity before commercialization. Often, an antibody is considered validated if it detects a band by Western blot of the expected molecular weight and, in some cases, if blocking peptides result in loss of staining. Neither of these approaches are unquestionable proof of target specificity. Since the oxytocin receptor has recently become a popular target in neuropsychiatric research, the need for specific antibodies to be used in brain has arisen. In this work, we have tested the specificity of six commercially available oxytocin receptor antibodies, indicated by the manufacturers to be suitable for Western blot and with an available image showing the correct size band (45-55 KDa). Antibodies were first tested by Western blot in brain lysates of wild-type and oxytocin receptor knockout mice. Uterus tissue was also tested as control for putative differential tissue specificity. In brain, the six tested antibodies lacked target specificity, as both wild-type and receptor knockout samples resulted in a similar staining pattern, including the expected 45-55 KDa band. Five of the six antibodies detected a selective band in uterus (which disappeared in knockout tissue). These five specific antibodies were also tested for immunohistochemistry in uterus, where only one was specific. However, when the uterine-specific antibody was tested in brain tissue, it lacked specificity. In conclusion, none of the six tested commercial antibodies are suitable to detect oxytocin receptor in brain by either Western blot or immunohistochemistry, although some do specifically detect it in uterus. The present work highlights the need to develop standardized antibody validation methods, including a proper negative control, in order to grant quality and reproducibility of the generated data.

Effect of antipsychotic drugs on group II metabotropic glutamate receptor expression and epigenetic control in postmortem brains of schizophrenia subjects.

Antipsychotic-induced low availability of group II metabotropic glutamate receptors (including mGlu2R and mGlu3R) in brains of schizophrenia patients may explain the limited efficacy of mGlu2/3R ligands in clinical trials. Studies evaluating mGlu2/3R levels in well-designed, large postmortem brain cohorts are needed to address this issue. Postmortem samples from the dorsolateral prefrontal cortex of 96 schizophrenia subjects and matched controls were collected. Toxicological analyses identified cases who were (AP+) or were not (AP-) receiving antipsychotic treatment near the time of death. Protein and mRNA levels of mGlu2R and mGlu3R, as well as GRM2 and GRM3 promoter-attached histone posttranslational modifications, were quantified. Experimental animal models were used to compare with data obtained in human tissues. Compared to matched controls, schizophrenia cortical samples had lower mGlu2R protein amounts, regardless of antipsychotic medication. Downregulation of mGlu3R was observed in AP- schizophrenia subjects only. Greater predicted occupancy values of dopamine D2 and serotonin 5HT2A receptors correlated with higher density of mGlu3R, but not mGlu2R. Clozapine treatment and maternal immune activation in rodents mimicked the mGlu2R, but not mGlu3R regulation observed in schizophrenia brains. mGlu2R and mGlu3R mRNA levels, and the epigenetic control mechanisms did not parallel the alterations at the protein level, and in some groups correlated inversely. Insufficient cortical availability of mGlu2R and mGlu3R may be associated with schizophrenia. Antipsychotic treatment may normalize mGlu3R, but not mGlu2R protein levels. A model in which epigenetic feedback mechanisms controlling mGlu3R expression are activated to counterbalance mGluR loss of function is described.

Maternal Immune Activation Induces Cortical Catecholaminergic Hypofunction and Cognitive Impairments in Offspring.

BACKGROUND: Impairment of specific cognitive domains in schizophrenia has been associated with prefrontal cortex (PFC) catecholaminergic deficits. Among other factors, prenatal exposure to infections represents an environmental risk factor for schizophrenia development in adulthood. However, it remains largely unknown whether the prenatal infection-induced changes in the brain may be associated with concrete switches in a particular neurochemical circuit, and therefore, if they could alter behavioral functions. METHODS: In vitro and in vivo neurochemical evaluation of the PFC catecholaminergic systems was performed in offspring from mice undergoing maternal immune activation (MIA). The cognitive status was also evaluated. Prenatal viral infection was mimicked by polyriboinosinic-polyribocytidylic acid (poly(I:C)) administration to pregnant dams (7.5 mg/kg i.p., gestational day 9.5) and consequences were evaluated in adult offspring. RESULTS: MIA-treated offspring showed disrupted recognition memory in the novel object recognition task (t = 2.30, p = 0.031). This poly(I:C)-based group displayed decreased extracellular dopamine (DA) concentrations compared to controls (t = 3.17, p = 0.0068). Potassium-evoked release of DA and noradrenaline (NA) were impaired in the poly(I:C) group (DA: Ft[10,90] = 43.33, p < 0.0001; Ftr[1,90] = 1.224, p = 0.2972; Fi[10,90] = 5.916, p < 0.0001; n = 11); (NA: Ft[10,90] = 36.27, p < 0.0001; Ftr[1,90] = 1.841, p = 0.208; Fi[10,90] = 8.686, p < 0.0001; n = 11). In the same way, amphetamine-evoked release of DA and NA were also impaired in the poly(I:C) group (DA: Ft[8,328] = 22.01, p < 0.0001; Ftr[1,328] = 4.507, p = 0.040; Fi[8,328] = 2.319, p = 0.020; n = 43); (NA: Ft[8,328] = 52.07; p < 0.0001; Ftr[1,328] = 4.322; p = 0.044; Fi[8,398] = 5.727; p < 0.0001; n = 43). This catecholamine imbalance was accompanied by increased dopamine D1 and D2 receptor expression (t = 2.64, p = 0.011 and t = 3.55, p = 0.0009; respectively), whereas tyrosine hydroxylase, DA and NA tissue content, DA and NA transporter (DAT/NET) expression and function were unaltered. CONCLUSIONS: MIA induces in offspring a presynaptic catecholaminergic hypofunction in PFC with cognitive impairment. This poly(I:C)-based model reproduces catecholamine phenotypes reported in schizophrenia and represents an opportunity for the study of cognitive impairment associated to this disorder.

The Matricellular Protein Hevin Is Involved in Alcohol Use Disorder.

Astrocytic-secreted matricellular proteins have been shown to influence various aspects of synaptic function. More recently, they have been found altered in animal models of psychiatric disorders such as drug addiction. Hevin (also known as Sparc-like 1) is a matricellular protein highly expressed in the adult brain that has been implicated in resilience to stress, suggesting a role in motivated behaviors. To address the possible role of hevin in drug addiction, we quantified its expression in human postmortem brains and in animal models of alcohol abuse. Hevin mRNA and protein expression were analyzed in the postmortem human brain of subjects with an antemortem diagnosis of alcohol use disorder (AUD, n = 25) and controls (n = 25). All the studied brain regions (prefrontal cortex, hippocampus, caudate nucleus and cerebellum) in AUD subjects showed an increase in hevin levels either at mRNA or/and protein levels. To test if this alteration was the result of alcohol exposure or indicative of a susceptibility factor to alcohol consumption, mice were exposed to different regimens of intraperitoneal alcohol administration. Hevin protein expression was increased in the nucleus accumbens after withdrawal followed by a ethanol challenge. The role of hevin in AUD was determined using an RNA interference strategy to downregulate hevin expression in nucleus accumbens astrocytes, which led to increased ethanol consumption. Additionally, ethanol challenge after withdrawal increased hevin levels in blood plasma. Altogether, these results support a novel role for hevin in the neurobiology of AUD.

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.

Characterization of Hevin (SPARCL1) Immunoreactivity in Postmortem Human Brain Homogenates.

Hevin is a matricellular glycoprotein that plays important roles in neural developmental processes such as neuronal migration, synaptogenesis and synaptic plasticity. In contrast to other matricellular proteins whose expression decreases when development is complete, hevin remains highly expressed, suggesting its involvement in adult brain function. In vitro studies have shown that hevin can have different post-translational modifications. However, the glycosylation pattern of hevin in the human brain remains unknown, as well as its relative distribution and localization. The present study provides the first thorough characterization of hevin protein expression by Western blot in postmortem adult human brain. Our results demonstrated two major specific immunoreactive bands for hevin: an intense band migrating around 130 kDa, and a band migrating around 100 kDa. Biochemical assays revealed that both hevin bands have a different glycosylation pattern. Subcellular fractionation showed greater expression in membrane-enriched fraction than in cytosolic preparation, and a higher expression in prefrontal cortex (PFC) compared to hippocampus (HIP), caudate nucleus (CAU) and cerebellum (CB). We confirmed that a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) and matrixmetalloproteinase 3 (MMP-3) proteases digestion led to an intense double band with similar molecular weight to that described as SPARC-like fragment (SLF). Finally, hevin immunoreactivity was also detected in human astrocytoma, meningioma, cerebrospinal fluid and serum samples, but was absent from any blood cell type.

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.

G Protein-Coupled Receptor Heteromers as Putative Pharmacotherapeutic Targets in Autism.

A major challenge in the development of pharmacotherapies for autism is the failure to identify pathophysiological mechanisms that could be targetable. The majority of developing strategies mainly aim at restoring the brain excitatory/inhibitory imbalance described in autism, by targeting glutamate or GABA receptors. Other neurotransmitter systems are critical for the fine-tuning of the brain excitation/inhibition balance. Among these, the dopaminergic, oxytocinergic, serotonergic, and cannabinoid systems have also been implicated in autism and thus represent putative therapeutic targets. One of the latest breakthroughs in pharmacology has been the discovery of G protein-coupled receptor (GPCR) oligomerization. GPCR heteromers are macromolecular complexes composed of at least two different receptors, with biochemical properties that differ from those of their individual components, leading to the activation of different cellular signaling pathways. Interestingly, heteromers of the above-mentioned neurotransmitter receptors have been described (e.g., mGlu2-5HT2A, mGlu5-D2-A2A, D2-OXT, CB1-D2, D2-5HT2A, D1-D2, D2-D3, and OXT-5HT2A). We hypothesize that differences in the GPCR interactome may underlie the etiology/pathophysiology of autism and could drive different treatment responses, as has already been suggested for other brain disorders such as schizophrenia. Targeting GPCR complexes instead of monomers represents a new order of biased agonism/antagonism that may potentially enhance the efficacy of future pharmacotherapies. Here, we present an overview of the crosstalk of the different GPCRs involved in autism and discuss current advances in pharmacological approaches targeting them.

Cartography of hevin-expressing cells in the adult brain reveals prominent expression in astrocytes and parvalbumin neurons.

Hevin, also known as SPARC-like 1, is a member of the secreted protein acidic and rich in cysteine family of matricellular proteins, which has been implicated in neuronal migration and synaptogenesis during development. Unlike previously characterized matricellular proteins, hevin remains strongly expressed in the adult brain in both astrocytes and neurons, but its precise pattern of expression is unknown. The present study provides the first systematic description of hevin mRNA distribution in the adult mouse brain. Using isotopic in situ hybridization, we showed that hevin is strongly expressed in the cortex, hippocampus, basal ganglia complex, diverse thalamic nuclei and brainstem motor nuclei. To identify the cellular phenotype of hevin-expressing cells, we used double fluorescent in situ hybridization in mouse and human adult brains. In the mouse, hevin mRNA was found in the majority of astrocytes but also in specific neuronal populations. Hevin was expressed in almost all parvalbumin-positive projection neurons and local interneurons. In addition, hevin mRNA was found in: (1) subsets of other inhibitory GABAergic neuronal subtypes, including calbindin, cholecystokinin, neuropeptide Y, and somatostatin-positive neurons; (2) subsets of glutamatergic neurons, identified by the expression of the vesicular glutamate transporters VGLUT1 and VGLUT2; and (3) the majority of cholinergic neurons from motor nuclei. Hevin mRNA was absent from all monoaminergic neurons and cholinergic neurons of the ascending pathway. A similar cellular profile of expression was observed in human, with expression of hevin in parvalbumin interneurons and astrocytes in the cortex and caudate nucleus as well as in cortical glutamatergic neurons. Furthermore, hevin transcript was enriched in ribosomes of astrocytes and parvalbumin neurons providing a direct evidence of hevin mRNAs translation in these cell types. This study reveals the unique and complex expression profile of the matricellular protein hevin in the adult brain. This distribution is compatible with a role of hevin in astrocytic-mediated adult synaptic plasticity and in the regulation of network activity mediated by parvalbumin-expressing neurons.

Oxytocin as Treatment for Social Cognition, Not There Yet.

In a short time, oxytocin has progressed from being a regular hormone involved in parturition and breastfeeding to be possibly the neuromodulator that has gathered the most attention. Attributed many positive roles in the modulation of different aspects of social behavior, such as bonding, empathy, cooperation, trust, and generosity, as well as roles as a natural anxiolytic and antidepressant, the expectations on oxytocin becoming a treatment for a number of disorders with associated social deficits have dramatically raised over the last years. However, despite the field has been investigating oxytocin's role in social behavior for over twenty years, there are still many unknowns on oxytocin's mechanisms of action and efficiency and the increasing number of clinical trials administering oxytocin to different clinical groups seem to disagree in its properties and report in most cases conflicting results. This has led to some disappointment among researchers and clinicians as oxytocin might not be the miraculous molecule that works in a "one size fits all" fashion initially considered. Conversely, this down-side of oxytocin might merely reflect the complexity of its neurotransmission system. The current reality is that, although oxytocin seems to have potential therapeutic value, there are key questions that remain unanswered as to decide the optimal target groups and treatment course. Here, we present an overview on critical points regarding the oxytocin system in health and disease that need to be better understood to establish its therapeutic properties and to decide who could benefit the most from its treatment.

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.

The Loss of α- and ß-Tubulin Proteins Are a Pathological Hallmark of Chronic Alcohol Consumption and Natural Brain Ageing.

Repetitive excessive alcohol intoxication leads to neuronal damage and brain shrinkage. We examined cytoskeletal protein expression in human post-mortem tissue from Brodmann's area 9 of the prefrontal cortex (PFC). Brain samples from 44 individuals were divided into equal groups of 11 control, 11 alcoholic, 11 non-alcoholic suicides, and 11 suicide alcoholics matched for age, sex, and post-mortem delay. Tissue from alcoholic cohorts displayed significantly reduced expression of α- and ß-tubulins, and increased levels of acetylated α-tubulin. Protein levels of histone deacetylase-6 (HDAC6), and the microtubule-associated proteins MAP-2 and MAP-tau were reduced in alcoholic cohorts, although for MAPs this was not significant. Tubulin gene expressions increased in alcoholic cohorts but not significantly. Brains from rats administered alcohol for 4 weeks also displayed significantly reduced tubulin protein levels and increased α-tubulin acetylation. PFC tissue from control subjects had reduced tubulin protein expression that was most notable from the sixth to the eighth decade of life. Collectively, loss of neuronal tubulin proteins are a hallmark of both chronic alcohol consumption and natural brain ageing. The reduction of cytosolic tubulin proteins could contribute to the brain volumetric losses reported for alcoholic patients and the elderly.

Characterisation of spinophilin immunoreactivity in postmortem human brain homogenates.

Spinophilin is a multifunctional scaffold protein that regulates the formation and function of dendritic spines and plays a role in neuronal migration. The distinct roles of spinophilin depend on its localization and the direct interaction with other proteins, which may target spinophilin to specific locations within the cell. Several studies suggest a role of spinophilin in the pathophysiology of neurological or psychiatric diseases. However, the majority have been performed in animals or cultured cells. Thus, the aim of the present study was to characterise the regional and subcellular expression of spinophilin immunoreactivity by western blot in postmortem human brain. Two specific immunoreactive bands for spinophilin were observed: an intense band migrating at around 120kDa, which seems to correspond to the apparent molecular weight of spinophilin described by other authors, and a less intense band of around 95kDa. This second form seems to be a proteolysis or cleavage product of the ~120kDa spinophilin. Interestingly, the subcellular distribution of both bands was different. In membrane fraction, the ~120kDa spinophilin band was the most abundant, whereas in cytosol it was the ~95kDa form. Furthermore, a different regional distribution for ~120kDa spinophilin band was observed, with the highest expression in prefrontal cortex, followed by hippocampus and cerebellum, and the lowest in caudate nucleus. Altogether, these results constitute a useful reference for future studies of spinophilin in pathological and non-pathological human brain tissues.

Structural and Functional Characterization of the Interaction of Snapin with the Dopamine Transporter: Differential Modulation of Psychostimulant Actions.

The importance of dopamine (DA) neurotransmission is emphasized by its direct implication in several neurological and psychiatric disorders. The DA transporter (DAT), target of psychostimulant drugs, is the key protein that regulates spatial and temporal activity of DA in the synaptic cleft via the rapid reuptake of DA into the presynaptic terminal. There is strong evidence suggesting that DAT-interacting proteins may have a role in its function and regulation. Performing a two-hybrid screening, we identified snapin, a SNARE-associated protein implicated in synaptic transmission, as a new binding partner of the carboxyl terminal of DAT. Our data show that snapin is a direct partner and regulator of DAT. First, we determined the domains required for this interaction in both proteins and characterized the DAT-snapin interface by generating a 3D model. Using different approaches, we demonstrated that (i) snapin is expressed in vivo in dopaminergic neurons along with DAT; (ii) both proteins colocalize in cultured cells and brain and, (iii) DAT and snapin are present in the same protein complex. Moreover, by functional studies we showed that snapin produces a significant decrease in DAT uptake activity. Finally, snapin downregulation in mice produces an increase in DAT levels and transport activity, hence increasing DA concentration and locomotor response to amphetamine. In conclusion, snapin/DAT interaction represents a direct link between exocytotic and reuptake mechanisms and is a potential target for DA transmission modulation.

Neuroprotective Effects of a Structurally New Family of High Affinity Imidazoline I2 Receptor Ligands.

The imidazoline I2 receptors (I2-IRs) are widely distributed in the brain, and I2-IR ligands may have therapeutic potential as neuroprotective agents. Since structural data for I2-IR remains unknown, the discovery of selective I2-IR ligands devoid of α2-adrenoceptor (α2-AR) affinity is likely to provide valuable tools in defining the pharmacological characterization of these receptors. We report the pharmacological characterization of a new family of (2-imidazolin-4-yl)phosphonates. Radioligand binding studies showed that they displayed a higher affinity for I2-IRs than idazoxan, and high I2/α2 selectivity. In vivo studies in mice showed that acute treatments with 1b and 2c significantly increased p-FADD/FADD ratio (an index of cell survival) in the hippocampus when compared with vehicle-treated controls. Additionally, acute and repeated treatments with 2c, but not with 1b, markedly reduced hippocampal p35 cleavage into neurotoxic p25. The present results indicate a neuroprotective potential of (2-imidazolin-4-yl)phosphonates acting at I2-IRs.

Altered CB1 receptor coupling to G-proteins in the post-mortem caudate nucleus and cerebellum of alcoholic subjects.

Biochemical, pharmacological and genetic evidence suggests the involvement of the endocannabinoid system in alcohol dependence. The aim of the present study was to evaluate the state of CB1 receptors in post-mortem caudate nucleus, hippocampus and cerebellum of alcoholic subjects.CB1 protein levels were measured by Western blot, CB1 receptor density and affinity by [(3)H]WIN55,212-2 saturation assays and CB1 functionality by [(35)S]GTPγS binding assays. Experiments were performed in samples from 24 subjects classified as non-suicidal alcoholics (n = 6), suicidal alcoholics (n = 6), non-alcoholic suicide victims (n = 6) and control subjects (n = 6).Alcoholic subjects presented hyperfunctional CB1 receptors in the caudate nucleus resulting in a higher maximal effect in both alcoholic groups compared to the non-alcoholic groups (p < 0.001). Conversely, in the cerebellum the non-suicidal alcoholic subjects showed hypofunctional receptors with lower maximal effect and potency (p < 0.001). No changes were found in the CB1 protein expression in either region. In the hippocampus of alcoholic subjects, no changes were observed either in the functionality, density or protein levels.Our data support an association between endocannabinoid system activity and alcoholism. The modifications reported here could be either a consequence of high lifetime ethanol consumption or a vulnerability factor to develop alcohol addiction.

Ctr9, a Protein in the Transcription Complex Paf1, Regulates Dopamine Transporter Activity at the Plasma Membrane.

Dopamine (DA) is a major regulator of sensorimotor and cognitive functions. The DA transporter (DAT) is the key protein that regulates the spatial and temporal activity of DA release into the synaptic cleft via the rapid reuptake of DA into presynaptic termini. Several lines of evidence have suggested that transporter-interacting proteins may play a role in DAT function and regulation. Here, we identified the tetratricopeptide repeat domain-containing protein Ctr9 as a novel DAT binding partner using a yeast two-hybrid system. We showed that Ctr9 is expressed in dopaminergic neurons and forms a stable complex with DAT in vivo via GST pulldown and co-immunoprecipitation assays. In mammalian cells co-expressing both proteins, Ctr9 partially colocalizes with DAT at the plasma membrane. This interaction between DAT and Ctr9 results in a dramatic enhancement of DAT-mediated DA uptake due to an increased number of DAT transporters at the plasma membrane. We determined that the binding of Ctr9 to DAT requires residues YKF in the first half of the DAT C terminus. In addition, we characterized Ctr9, providing new insight into this protein. Using three-dimensional modeling, we identified three novel tetratricopeptide repeat domains in the Ctr9 sequence, and based on deletion mutation experiments, we demonstrated the role of the SH2 domain of Ctr9 in nuclear localization. Our results demonstrate that Ctr9 localization is not restricted to the nucleus, as previously described for the transcription complex Paf1. Taken together, our data provide evidence that Ctr9 modulates DAT function by regulating its trafficking.

Isoaspartate, carbamoyl phosphate synthase-1, and carbonic anhydrase-III as biomarkers of liver injury.

We had previously shown that alcohol consumption can induce cellular isoaspartate protein damage via an impairment of the activity of protein isoaspartyl methyltransferase (PIMT), an enzyme that triggers repair of isoaspartate protein damage. To further investigate the mechanism of isoaspartate accumulation, hepatocytes cultured from control or 4-week ethanol-fed rats were incubated in vitro with tubercidin or adenosine. Both these agents, known to elevate intracellular S-adenosylhomocysteine levels, increased cellular isoaspartate damage over that recorded following ethanol consumption in vivo. Increased isoaspartate damage was attenuated by treatment with betaine. To characterize isoaspartate-damaged proteins that accumulate after ethanol administration, rat liver cytosolic proteins were methylated using exogenous PIMT and (3)H-S-adenosylmethionine and proteins resolved by gel electrophoresis. Three major protein bands of ∼ 75-80 kDa, ∼ 95-100 kDa, and ∼ 155-160 kDa were identified by autoradiography. Column chromatography used to enrich isoaspartate-damaged proteins indicated that damaged proteins from ethanol-fed rats were similar to those that accrued in the livers of PIMT knockout (KO) mice. Carbamoyl phosphate synthase-1 (CPS-1) was partially purified and identified as the ∼ 160 kDa protein target of PIMT in ethanol-fed rats and in PIMT KO mice. Analysis of the liver proteome of 4-week ethanol-fed rats and PIMT KO mice demonstrated elevated cytosolic CPS-1 and betaine homocysteine S-methyltransferase-1 when compared to their respective controls, and a significant reduction of carbonic anhydrase-III (CA-III) evident only in ethanol-fed rats. Ethanol feeding of rats for 8 weeks resulted in a larger (∼ 2.3-fold) increase in CPS-1 levels compared to 4-week ethanol feeding indicating that CPS-1 accumulation correlated with the duration of ethanol consumption. Collectively, our results suggest that elevated isoaspartate and CPS-1, and reduced CA-III levels could serve as biomarkers of hepatocellular injury.

The endocannabinoid system is altered in the post-mortem prefrontal cortex of alcoholic subjects.

There is strong biochemical, pharmacological and genetic evidence for the involvement of the endocannabinoid system (ECS) in alcohol dependence. However, the majority of studies have been performed in animal models. The aim of the present study was to assess the state of the CB1 receptor, the enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), and the extracellular signal-regulated kinase (ERK) and cyclic-AMP response element-binding protein (CREB) in the post-mortem prefrontal cortex of alcoholic subjects. Experiments were performed in samples from 44 subjects classified in four experimental groups: (1) non-suicidal alcoholic subjects (n = 11); (2) suicidal alcoholic subjects (n = 11); (3) non-alcoholic suicide victims (n = 11); and (4) control subjects (n = 11). We did not observe statistically significant differences in CB1 mRNA relative expression among the four experimental groups. Conversely, our results showed an increase in CB1 receptor protein expression in the prefrontal cortex of the suicidal alcoholic group (127.2 ± 7.3%), with no changes in functionality with regard to either G protein activation or the inhibition of adenylyl cyclase. In parallel, alcoholic subjects presented lower levels of MAGL activity, regardless of the cause of death. A significant decrease in the active form of ERK and CREB levels was also observed in both alcoholic groups. Taken together, our data are consistent with a role for the ECS in the neurobiological mechanisms underlying alcoholism. Moreover, the alterations reported here should be of great interest for the therapeutic treatment of this chronic psychiatric disease.

Alcohol-related brain damage in humans.

Chronic excessive alcohol intoxications evoke cumulative damage to tissues and organs. We examined prefrontal cortex (Brodmann's area (BA) 9) from 20 human alcoholics and 20 age, gender, and postmortem delay matched control subjects. H & E staining and light microscopy of prefrontal cortex tissue revealed a reduction in the levels of cytoskeleton surrounding the nuclei of cortical and subcortical neurons, and a disruption of subcortical neuron patterning in alcoholic subjects. BA 9 tissue homogenisation and one dimensional polyacrylamide gel electrophoresis (PAGE) proteomics of cytosolic proteins identified dramatic reductions in the protein levels of spectrin ß II, and α- and ß-tubulins in alcoholics, and these were validated and quantitated by Western blotting. We detected a significant increase in α-tubulin acetylation in alcoholics, a non-significant increase in isoaspartate protein damage, but a significant increase in protein isoaspartyl methyltransferase protein levels, the enzyme that triggers isoaspartate damage repair in vivo. There was also a significant reduction in proteasome activity in alcoholics. One dimensional PAGE of membrane-enriched fractions detected a reduction in ß-spectrin protein levels, and a significant increase in transmembranous α3 (catalytic) subunit of the Na+,K+-ATPase in alcoholic subjects. However, control subjects retained stable oligomeric forms of α-subunit that were diminished in alcoholics. In alcoholics, significant loss of cytosolic α- and ß-tubulins were also seen in caudate nucleus, hippocampus and cerebellum, but to different levels, indicative of brain regional susceptibility to alcohol-related damage. Collectively, these protein changes provide a molecular basis for some of the neuronal and behavioural abnormalities attributed to alcoholics.