Regulation of alpha7-nicotinic receptor subunit and alpha7-like gene expression in the prefrontal cortex of patients with bipolar disorder and schizophrenia.

OBJECTIVE: The alpha7-nicotinic receptor subunit gene (CHRNA7) is located at chromosome 15q13-14, a region previously linked with schizophrenia. Genetic association and mRNA expression studies also implicate CHRNA7 in schizophrenia. The CHRNA7 gene has a partial duplication that constitutes the alpha7-like nicotinic receptor gene (CHRFAM7A). We hypothesized that major psychoses could affect the expression of both CHRNA7 and CHRFAM7A. METHOD: CHRNA7 and CHRFAM7A mRNA levels were measured in postmortem prefrontal cortex (donated by the Stanley Foundation) from subjects with schizophrenia, bipolar disorder and unaffected controls (n = 35 each). RESULTS: The mRNA levels of alpha7 and alpha7-like genes have a positive correlation overall (r = 0.25; P = 0.009), however, there is no significant difference in the expression of CHRNA7 among the three diagnostic groups. CONCLUSION: This correlation is driven by the bipolar group (r = 0.43; P = 0.009), and is absent in schizophrenia and unaffected controls, suggesting an alteration in the CHRNA7:CHRFAM7A ratio in bipolar disorder.

The interaction between TPH2 promoter haplotypes and clinical-demographic risk factors in suicide victims with major psychoses.

Tryptophan hydroxylase isoform 2 (TPH2) is a rate-limiting enzyme in the biosynthesis of serotonin (5-HT) and is predominantly localized in the brain. Previous studies have suggested that there is an association between serotonergic dysfunction in the brain and suicidality. This study was designed to examine whether the -473T > A and -8396G > C polymorphisms of the TPH2 gene may be associated with completed suicide in subjects with major psychoses from the Stanley Foundation Brain Bank sample. TPH2 genotypes were determined in 69 subjects with a diagnosis of schizophrenia or bipolar disorder, among which 22 died by suicide. Genomic DNA was amplified by polymerase chain reaction and typed by automated methods. Both markers were found to be in Hardy-Weinberg equilibrium and in strong linkage disequilibrium. No association with history of suicide was found for either polymorphism. Haplotype analysis with EHAP showed no association between completed suicide and haplotype distribution (chi2 = 1.877; 3 df; P = 0.598). Nor was there any association between suicide and these genetic markers even when clinical-demographic factors were considered as covariates in the haplotype analysis. These findings suggest that these 5' marker haplotypes in the TPH2 gene do not influence suicidal behaviour.

The human dopamine D4 receptor repeat sequences modulate expression.

Genetic studies have implicated a polymorphic repeat sequence in exon 3 of the human dopamine D4 receptor in various behavioral and psychiatric disorders. Functionally various repeat variants are nearly identical, but whether these have different effects on gene expression has not been studied. To study the role of the repeat sequences on expression independently from its structural and functional effects at the protein level, we introduced these sequences immediately upstream of the promoter and in the 3' untranslated region of a luciferase reporter vector. In this report, we demonstrate that the repeat sequence can both modulate promoter activity and alter expression post-transcriptionally. The repeat sequence can serve as a substrate for a nuclear binding factor and all the three repeat variants can suppress promoter activity. Placement of the three repeat variants downstream from the luciferase gene in the expression vector shows, however, that the D4.7 repeat sequence has significantly suppressed expression of the reporter compared to the D4.2 and D4.4 repeats, likely via mechanisms involving RNA stability or translational efficiency. These data indicate that the various D4 repeat sequences have different effects on expression, which may explain its potential role in behavioral disorders.

Identification of candidate genes for psychosis in rat models, and possible association between schizophrenia and the 14-3-3eta gene.

Although the genetic contribution to schizophrenia is substantial, positive findings in whole-genome linkage scans have not been consistently replicated. We analyzed gene expression in various rat conditions to identify novel candidate genes for schizophrenia. Suppression subtraction hybridization (SSH), with polyA mRNA from temporal and frontal cortex of rats, was used to identify differentially expressed genes. Expression of mRNA was compared between adult Lewis and Fischer 344 (F344) rats, adult and postnatal day 6 (d6) F344, and adult F344 treated with haloperidol or control vehicle. These groups were chosen because each highlights a particular aspect of schizophrenia: differences in strain vulnerability to behavioral analogs of psychosis; factors that may relate to disease onset in relation to CNS development; and improvement of symptoms by haloperidol. The 14-3-3 gene family, as represented by 14-3-3gamma and 14-3-3zeta isoforms in the SSH study, and SNAP-25 were among the candidate genes. Genetic association between schizophrenia and the 14-3-3eta gene, positioned close to a genomic locus implicated in schizophrenia, and SNAP-25 genes was analyzed in 168 schizophrenia probands and their families. These findings address three different genes in the 14-3-3 family. We find a significant association with schizophrenia for two polymorphisms in the 14-3-3eta gene: a 7 bp variable number of tandem repeats in the 5' noncoding region (P=0.036, 1 df), and a 3' untranslated region SNP (753G/A) that is an RFLP visualized with Ava II (P=0.028). There was no significant genetic association with SNAP-25. The candidate genes identified may be of functional importance in the etiology, pathophysiology or treatment response of schizophrenia or psychotic symptoms. This is to our knowledge the first report of a significant association between the 14-3-3eta-chain gene and schizophrenia in a family-based sample, strengthening prior association reports in case-control studies and microarray gene expression studies.

SH3 ligands in the dopamine D3 receptor.

It has recently been observed that G protein-coupled receptors (GPCRs) can interact with SH3 domains through polyproline motifs. These interactions appear to be involved in receptor internalization and MAPK signalling. Here we report that the third cytoplasmic loop of the dopamine D3 receptor can interact in vitro with the adaptor protein Grb2. While the amino- and carboxy-terminal SH3 domains of Grb2 separately did not interact with the D3 receptor loop, the interaction is at least partially maintained with a Grb2 mutant for the amino-terminal SH3 domain, but disrupted for a Grb2 mutant with a nonfunctional carboxy-terminal SH3 domain. The data indicate the need of structural integrity of the entire Grb2 protein for the interaction and dominant role of the carboxy-terminal SH3 domain in the interaction. Disruption of the PXXP motifs in the D3 receptor did not affect the interaction with Grb2. These results indicate that GPCRs may contain SH3 ligands that do not contain the postulated minimal consensus sequence PXXP.

Nature of the oligomers formed by muscarinic m2 acetylcholine receptors in Sf9 cells.

Wild-type, FLAG-tagged, and c-myc-tagged muscarinic m2 receptors extracted in digitonin-cholate from singly and co-infected Sf9 (Spodoptera frugiperda) cells were indistinguishable in their binding of [3H]quinuclidinylbenzilate, either before or after purification. The FLAG epitope was found to coimmunoprecipitate with the c-myc epitope when co-infected cells were solubilised in digitonin-cholate, n-dodecyl-beta-D-maltoside or Lubrol-PX. The degree of coprecipitation in digitonin-cholate was unaffected by preincubation of the extract for up to 60 min at 30 degrees C, with or without muscarinic receptor ligands; no coimmunoprecipitation occurred in mixed extracts from singly infected cells. As measured by [3H]quinuclidinylbenzilate, the efficiency of immunoprecipitation from co-infected cells was 87% of that from singly infected cells. The amount of receptor immunoprecipitated from the latter, as determined by densitometry, was 2.3-fold that expected from the loss of binding from the extract. The data suggest that at least some of the receptors were trimeric or larger and that oligomers neither formed nor dissociated under the conditions of the experiments. Also, some receptors appear to be non-functional or latent in digitonin-solubilised extracts.

Dopamine D(4) and D(2L) Receptor Stimulation of the Mitogen-Activated Protein Kinase Pathway Is Dependent on trans-Activation of the Platelet-Derived Growth Factor Receptor.

The ability of dopamine D(4) and D(2) receptors to activate extracellular signal-regulated kinases (ERKs) 1 and 2 was compared using Chinese hamster ovary (CHO-K1) cells transfected with D(4.2), D(4.4), D(4.7), and D(2L) receptors. Dopamine stimulation of D(4) or D(2L) receptors produced a transient, dose-dependent increase in ERK1/2 activity. Receptor-specific activation of the ERK mitogen-activated protein kinase (MAPK) pathway was confirmed using the D(2)-like receptor-selective agonist quinpirole, whereas the specific antagonist haloperidol blocked activation. MAPK stimulation was dependent on a pertussis-toxin-sensitive G protein (G(i/o)). trans-Activation of the platelet-derived growth factor (PDGF) receptor was an essential step in D(4) and D(2L) receptor-induced MAPK activation. PDGF receptor-selective tyrosine kinase inhibitors tyrphostin A9 and AG1295 abolished or significantly inhibited ERK1/2 activation by D(4) and D(2L) receptors. Dopamine stimulation of the D(4) receptor also produced a rapid increase in tyrosine phosphorylation of the PDGF receptor-beta. The Src-family tyrosine kinase inhibitor PP2 blocked MAPK activation by dopamine; however, this drug was also found to inhibit PDGF-BB-stimulated ERK activity and autophosphorylation of the PDGF receptor-beta. Downstream signaling pathways support the involvement of a receptor tyrosine kinase. The phosphoinositide 3-kinase inhibitors wortmannin and LY294002, protein kinase C inhibitors GF109203X and Calphostin C, dominant-negative RasN17, and the MEK inhibitor PD98059 significantly attenuated or abolished activation of MAPK by dopamine D(4) and D(2L) receptors. Our results indicate that D(4) and D(2L) receptors activate the ERK kinase cascade by first mobilizing signaling by the PDGF receptor, followed by the subsequent activation of ERK1/2 by pathways associated with this receptor tyrosine kinase.

Dopamine signaling in Caenorhabditis elegans-potential for parkinsonism research.

The nematode Caenorhabditis elegans is an attractive model system for the study of many biological processes. It possesses a simple nervous system with known anatomy and connectivity, is conveniently and cheaply cultured in the laboratory, and is amenable to many genetic manipulations that are impossible in mammalian systems. The recent completion of the C. elegans genome sequence provides a rich resource of genomic and bioinformatic data to researchers in diverse fields. This organism, however, has been underexploited in the studies of many basic processes related to nervous system function, neuropsychiatric disorders and neuromuscular function. Anatomical, biochemical, behavioral, pharmacological and genetic evidence accumulated to date strongly suggests that dopamine is used as a neurotransmitter by C. elegans, and that its effects are mediated through pathway(s) that share many features with those of mammals. DNA sequence analysis reveals genes highly homologous to those encoding mammalian dopamine receptors. Probably, C. elegans has dopamine receptors that transduce environmental cues into behaviors, and these receptors pharmacologically most closely resemble the D2 family. Here we present a review of the current state of research into the dopamine system of the worm, focussing on its potential for use in the study of biological processes related to parkinsonism.

The dopamine D(4) receptor: one decade of research.

Dopamine is an important neurotransmitter involved in motor control, endocrine function, reward, cognition and emotion. Dopamine receptors belong to the superfamily of G protein-coupled receptors and play a crucial role in mediating the diverse effects of dopamine in the central nervous system (CNS). The dopaminergic system is implicated in disorders such as Parkinson's disease and addiction, and is the major target for antipsychotic medication in the treatment of schizophrenia. Molecular cloning studies a decade ago revealed the existence of five different dopamine receptor subtypes in mammalian species. While the presence of the abundantly expressed dopamine D(1) and D(2) receptors was predicted from biochemical and pharmacological work, the cloning of the less abundant dopamine D(3), D(4) and D(5) receptors was not anticipated. The identification of these novel dopamine receptor family members posed a challenge with respect to determining their precise physiological roles and identifying their potential as therapeutic targets for dopamine-related disorders. This review is focused on the accomplishments of one decade of research on the dopamine D(4) receptor. New insights into the biochemistry of the dopamine D(4) receptor include the discovery that this G protein-coupled receptor can directly interact with SH3 domains. At the physiological level, converging evidence from transgenic mouse work and human genetic studies suggests that this receptor has a role in exploratory behavior and as a genetic susceptibility factor for attention deficit hyperactivity disorder.

Dopamine receptor gene transfer into rat striatum using a recombinant adenoviral vector: rotational behaviour.

To study the role of dopamine (DA) receptor expression on dopamine-mediated rotational behaviour, adenovirus expressing the lacZ reporter gene (AdCMVLacZ) or D2R expressing adenoviral vector (AdRSVD2) viruses, mediating expression of beta-galactosidase and DAD2 receptors, respectively, were microinjected stereotactically into Sprague-Dawley rat striatum. Apomorphine stimulated rotational behaviour was measured in rats unilaterally injected with either AdCMVLacZ or AdRSVD2. No significant difference in rotational direction was observed until day 14 post-injection, when animals showed a tendency to rotate away from the injected side. Our data indicate that unilateral changes in receptor density mediated by a non-cell type selective adenoviral vector results in minor changes in rotational behavior. This suggests that supersensitivity in dopamine receptor signaling, rather than receptor levels per se, are the major factor in determining rotational response with dopamine agonist stimulation in unilateral striatal dopamine depleted animals.

Comparative pharmacological and functional analysis of the human dopamine D4.2 and D4.10 receptor variants.

The human dopamine D4 receptor is a D2-like receptor which is a target for most common neuroleptics. Previous investigations have shown that this receptor displays a large polymorphic variation in the third intracellular loop involving a variable number of direct imperfect tandem repeats (VNTR) of 16 amino acids. The shortest and longest repeat variants reported to date contain two and 10 repeat units (D4.2 and D4.10). No major pharmacological differences have been reported for the most common variants of this receptor (D4.2, D4.4 and D4.7), although the D4.7 was reported by us to display a slightly lower potency for dopamine in functional assays. Direct pharmacological and functional comparison of the longest and shortest variants in this study suggest no major discrepancies in pharmacological or functional profile between both receptors. Both receptors display, on average, a 15-fold and 90-fold lower potency for epinephrine and norepinephrine, respectively, compared with dopamine. We observed small increases in functional potency and affinity for dopamine and quinpirole at the D4.10 receptor variant compared with the D4.2 receptor. Our data indicate that there is no direct relationship between the length of the polymorphism and changes in pharmacology or functional activity. These findings are a suitable caution against the arbitrary pooling of D4 receptor VNTR genotypes in genetic studies, based on length.

Short- and long-term heterologous sensitization of adenylate cyclase by D4 dopamine receptors.

The D4 dopamine receptor, a member of the D2-like dopamine receptor family, may be important in the etiology and treatment of schizophrenia. The present study was designed to examine the effects of dopamine agonist exposure on adenylate cyclase activity in HEK293 cells stably expressing recombinant-D4 receptors. Two hour pretreatment with dopamine receptor agonists resulted in heterologous sensitization of forskolin-stimulated cyclic AMP accumulation in intact cells expressing the D4.2, D4.4, or D4.7 dopamine receptor variant. The potency and efficacy of dopamine for sensitization of cyclic AMP accumulation was comparable at all D4 receptor variants. D4 dopamine receptor-mediated sensitization was blocked by the D4 antagonist, clozapine, and prevented by overnight pretreatment with pertussis toxin, implying a role for Gi/Go proteins in heterologous sensitization. Further, long-term (18 h) agonist exposure resulted in a greater degree of sensitization of forskolin-stimulated cyclic AMP accumulation in both intact cells and membrane preparations of cells expressing the D4 receptor, compared to 2 h agonist exposure, without altering the density of the receptors. In addition, long-term agonist exposure decreased the abundance of Gialpha without altering the abundance of Gsalpha, whereas short-term agonist treatment had no effect on the immunoreactivity of either G protein. In summary, long-term agonist-induced sensitization of adenylate cyclase by the D4 receptor may involve mechanisms that do not contribute to short-term sensitization.

Co-expression of human Kir3 subunits can yield channels with different functional properties.

To date, no comprehensive study has been done on all combinations of the human homologues of the Kir3.0 channel family, and the human homologue of Kir3.3 has not yet been identified. To obtain support for the contention that most of the functional data on non-human Kir3.0 channels can be extrapolated to human channels, we have cloned the human homologues of the Kir3.0 family, including the yet unidentified human Kir3.3, and the human Kir4.1. The expression pattern of these channels in various human brain areas and peripheral tissues, analysed by Northern blot analysis, allows for the existence of various homomeric and heteromeric forms of human Kir3.0 channels. Expression studies of all possible combinations in Xenopus oocytes indicated that in homomeric Kir3.2c and heteromeric Kir3.1/3.2c channels mediate, in our studies, inward currents with largest amplitude of any other Kir3.0 channel combinations, followed by heteromeric Kir3.1/3.4 and homomeric Kir4.1 channels. Channel combinations which include Kir3.3 are detrimental to the formation of functional channels. The co-expression experiments with different Kir channel subunits indicate the selective formation of certain channel combinations, suggesting that channel specificity is not solely dependent on spatial and temporal regulation of Kir subunit expression.

SH3 binding domains in the dopamine D4 receptor.

The dopamine D4 receptor is a G protein-coupled receptor (GPCR) that belongs to the dopamine D2-like receptor family. Functionally, the D2-like receptors are characterized by their ability to inhibit adenylyl cyclase. The dopamine D4 receptor as well as many other catecholaminergic receptors contain several putative SH3 binding domains. Most of these sites in the D4 receptor are located in a polymorphic repeat sequence and flanking sequences in the third intracellular loop. Here we demonstrate that this region of the D4 receptor can interact with a large variety of SH3 domains of different origin. The strongest interactions were seen with the SH2-SH3 adapter proteins Grb2 and Nck. The repeat sequence itself is not essential in this interaction. The data presented indicate that the different SH3 domains in the adapter proteins interact in a cooperative fashion with two distinct sites immediately upstream and downstream from the repeat sequence. Removal of all the putative SH3 binding domains in the third intracellular loop of the dopamine D4 receptor resulted in a receptor that could still bind spiperone and dopamine. Dopamine could not modulate the coupling of these mutant receptors to adenylyl cyclase and MAPK, although dopamine modulated receptor-G protein interaction appeared normal. The receptor deletion mutants show strong constitutive internalization that may account for the deficiency in functional activation of second messengers. The data indicates that the D4 receptor contains SH3 binding sites and that these sites fall within a region involved in the control of receptor internalization.

Promoter-independent regulation of cell-specific dopamine receptor expression.

Here we describe the construction of recombinant adenoviruses expressing dopamine D2 and D4 receptors, and their ability to mediate high levels of heterologous expression in a variety of cell types in vitro and in vivo for at least 7 days post infection. These experiments demonstrated that maximum receptor expression is achieved generally within 24 h and remains constant thereafter. Maximum expression levels were highly variable between cell lines and dependent on infection efficiency and promoter strength. Correction for these two variables revealed differences in relative expression levels between cell lines varying by two orders of magnitude. Our results indicate that in addition to gene transcription, post-transcriptional mechanisms play a dominant role in determining dopamine receptor levels in this system.

Dopamine D2 and D4 receptor ligands: relation to antipsychotic action.

Since the discovery that the antipsychotic action of phenothiazines was mediated by dopamine D2 receptors, the dopamine system has been scrutinized for schizophrenia related abnormalities. The focus has been to create neuroleptics with improved antipsychotic profiles and reduced side effects. With the identification of multiple dopamine receptor subtypes, the hypotheses regarding the role of dopamine in schizophrenia and antipsychotic action of neuroleptics have been refined. Even after the molecular identification of newer dopamine D2-like receptor subtypes (D3 and D4), the dopamine D2 receptor is still considered the predominant site for antipsychotic action. However, there has been much debate concerning the modulatory role of other dopamine receptor sites in the mechanism of action of antipsychotic drugs. Specifically, the dopamine D4 receptor has received much attention in this regard, since the atypical antipsychotic agent, clozapine, preferentially blocks this receptor subtype as compared with dopamine D2 and D3 receptors. In this review we will highlight some of the observations and arguments regarding the involvement of the dopamine D2 and D4 receptor sites in the therapeutic efficacy of antipsychotic medication.

Dopamine D4 receptor-mediated inhibition of cyclic adenosine 3',5'-monophosphate production does not affect prolactin regulation.

Under physiological conditions, PRL synthesis and secretion are predominantly under negative control by dopamine acting through dopamine D2 receptors present in the pituitary lactotroph cells. To investigate the role of D4 receptors in the regulation of PRL synthesis and secretion, we stably transfected the human D4 receptor complementary DNA into the somatomammotrophic cell line GH4C1. The pharmacological characteristics of D4 expressed in GH4C1 were in close agreement with previous D4 receptor studies in Chinese hamster ovary and COS-7 cells. In GH4C1 cells, activation of D4 receptor variants (D4.2, D4.4, and D4.7) resulted in a similar level of reduction in forskolin- and vasoactive intestinal peptide (VIP)-stimulated cAMP levels (33% and 50%, respectively). In addition, the forskolin-stimulated activity of cAMP response elements fused to the VIP promoter driving the lacZ reporter gene could be blocked by D4 activation. However, quinpirole treatment had a minimal effect on transiently expressed luciferase reporter gene driven by a proximal PRL promoter in one of the D4-expressing cell lines. In contrast, the dopamine D2short receptor expressing GH4ZR7 cells treated with quinpirole displayed a significant decrease (51.3 +/- 4.1%) in PRL promoter activity. VIP-stimulated PRL release was not affected by D4 receptor activation, whereas in GH4ZR7 cells, a significant decrease in VIP-stimulated PRL levels was observed. Neither PRL promoter activity nor PRL secretion levels were affected in control untransfected GH4C1 cells. From this study it appears that although the D4 receptor may be expressed in the anterior pituitary, it does not have a major effect on PRL promoter activity or PRL secretion in GH4C1 cells despite its ability to reduce cAMP production. This might explain why D4- over D2-preferring antipsychotics such as clozapine do not cause hyperprolactinemia.

Review the role of dopamine D4 receptors in schizophrenia and antipsychotic action.

For the past 20 years the most enduring explanation for schizophrenia has been the dopamine hypothesis, which proposes that the dopaminergic system is overactive in this widespread disease. Classically, the D2 receptor formed the core of the dopamine hypothesis since there was considerable evidence for elevations of D2 receptor levels in the brains of schizophrenic patients, and because these receptors served as the primary target in mediating antipsychotic effects of most neuroleptics. However, the dopamine D4 receptor has recently received particular attention in this context. This is because the atypical antipsychotic, clozapine, which is effective in treating refractory schizophrenics without the side-effect profile of typical neuroleptics, displays a 10-fold higher affinity for D4 compared to D2 or D3 receptors. Furthermore, the concentration in plasma water of clinical doses ofclozapine correlates well with its in vitro binding affinity for D4, but not D2 or D3 receptors, suggesting that D4 is a potential target in mediating clozapine's antipsychotic effects. As well, marked elevations in the level of a D4-like site (not identical to the D4 receptor) has been seen in the striatum of postmortem schizophrenic brains, but not in control brains. Finally, the most interesting feature of the D4 receptor is perhaps the array of polymorphisms associated with it, creating structural diversity in this receptor that supercedes all other known catecholamine receptors. The existence of these D4 polymorphisms raises the possibility that structural variations of this receptor may be associated with an increased susceptibility to schizophrenia, or observed variations in individual response to clozapine treatment. However, several studies aimed at investigating these hypotheses could not establish a direct role of D4 in schizophrenia. Furthermore, no association was evident between the polymorphic forms of D4 and susceptibility to schizophrenia, or variable clozapine response. Nevertheless, investigations surrounding this receptor has been far from futile. The observations which support the idea that D4 might serve as a target for clozapine have significantly modified and extended our understanding of mechanisms underlying atypical antipsychotic treatment of schizophrenia, as well as the dopamine hypothesis for schizophrenia. Further characterization of this receptor may prove to be crucial in designing highly effective antipsychotic drugs with minimal contraindications.