GPR37 and GPR37L1 differently interact with dopamine 2 receptors in live cells.

Receptor-receptor interactions are essential to fine tune receptor responses and new techniques enable closer characterization of the interactions between involved proteins directly in the plasma membrane. Fluorescence cross-correlation spectroscopy (FCCS), which analyses concurrent movement of bound molecules with single-molecule detection limit, was here used to, in live N2a cells, study interactions between the Parkinson's disease (PD) associated orphan receptor GPR37, its homologue GPR37L1, and the two splice variants of the dopamine 2 receptor (D2R). An interaction between GPR37 and both splice forms of D2R was detected. 4-phenylbutyrate (4-PBA), a neuroprotective chemical chaperone known to increase GPR37 expression at the cell surface, increased the fraction of interacting molecules. The interaction was also increased by pramipexole, a D2R agonist commonly used in the treatment of PD, indicating a possible clinically relevance. Cross-correlation, indicating interaction between GPR37L1 and the short isoform of D2R, was also detected. However, this interaction was not changed with 4-PBA or pramipexole treatment. Overall, these data provide further evidence that heteromeric GPR37-D2R exist and can be pharmacologically modulated, which is relevant for the treatment of PD. This article is part of the Special Issue entitled 'Receptor heteromers and their allosteric receptor-receptor interactions'.

Genes and alcohol.

The negative effects of excessive alcohol use include dependence, psychiatric co-morbidity and increased risk for suicide. A dominating risk factor is heritage. A large number of studies have addressed the genetic basis, either "candidate genes" in the brain reward system, or searched for unknown genes in family studies by linkage analysis. It is clear that no single gene polymorphism is of use in preventive medicine. A consistent finding, however, is that polymorphism in the alcohol dehydrogenase cluster and other metabolic pathways are of some relevance on a population basis, suggesting a link between alcohol toxicity in general and dependence. An emerging concern is potential gender differences as women, who are generally more sensitive, acquire male drinking habits.

The 2009 Nobel conference on the role of genetics in promoting suicide prevention and the mental health of the population.

A 3-day Nobel Conference entitled 'The role of genetics in promoting suicide prevention and the mental health of the population' was held at the Nobel Forum, Karolinska Institute (KI) in Stockholm, Sweden, during 8-10 June 2009. The conference was sponsored by the Nobel Assembly for Physiology or Medicine and organized by the National Prevention for Suicide and Mental Ill-Health and the Center for Molecular Medicine at KI. The program consisted of 19 invited presentations, covering the genetic basis of mood/psychotic disorders and substance abuse in relation to suicide, with topics ranging from cellular-molecular mechanisms to (endo)phenotypes of mental disorders at the level of the individual and populations. Here, we provide an overview based on the highlights of what was presented.

Analysis of 10 independent samples provides evidence for association between schizophrenia and a SNP flanking fibroblast growth factor receptor 2.

We and others have previously reported linkage to schizophrenia on chromosome 10q25-q26 but, to date, a susceptibility gene in the region has not been identified. We examined data from 3606 single-nucleotide polymorphisms (SNPs) mapping to 10q25-q26 that had been typed in a genome-wide association study (GWAS) of schizophrenia (479 UK cases/2937 controls). SNPs with P<0.01 (n=40) were genotyped in an additional 163 UK cases and those markers that remained nominally significant at P<0.01 (n=22) were genotyped in replication samples from Ireland, Germany and Bulgaria consisting of a total of 1664 cases with schizophrenia and 3541 controls. Only one SNP, rs17101921, was nominally significant after meta-analyses across the replication samples and this was genotyped in an additional six samples from the United States/Australia, Germany, China, Japan, Israel and Sweden (n=5142 cases/6561 controls). Across all replication samples, the allele at rs17101921 that was associated in the GWAS showed evidence for association independent of the original data (OR 1.17 (95% CI 1.06-1.29), P=0.0009). The SNP maps 85 kb from the nearest gene encoding fibroblast growth factor receptor 2 (FGFR2) making this a potential susceptibility gene for schizophrenia.

Regional adaptations in PSD-95, NGFI-A and secretogranin gene transcripts related to vulnerability to behavioral sensitization to amphetamine in the Roman rat strains.

Genetically selected for high or low two-way active avoidance, Roman high-avoidance (RHA) and Roman low-avoidance (RLA) rats differ in their central dopaminergic activity, sensation/novelty- and substance-seeking profiles. These animals are, therefore, well suited to identify anatomical and neurochemical concomitants of behavioral sensitization, a phenomenon linked to addictive liability. We submitted inbred RHA (RHA-I), inbred RLA (RLA-I) and Sprague-Dawley-OFA (SD-OFA) rats to a sensitization regimen with amphetamine and studied the behavioral response to an amphetamine challenge after a 2-week withdrawal period. The expression patterns of nerve growth factor inducible clone A (NGFI-A), secretogranin, post-synaptic density protein of 95 Kd (PSD-95), prodynorphin and proenkephalin mRNA were also analyzed using in situ hybridization, after the challenge with amphetamine. RHA-I rats showed stronger sensitization than SD-OFA rats. RLA-I rats did not show sensitization but were hyper-reactive to amphetamine. Expression of behavioral sensitization in RHA-I rats activated secretogranin and PSD-95 mRNA in the nucleus accumbens core. On the other hand, high induction of NGFI-A mRNA in the central amygdala was observed in RLA-I rats when they experienced amphetamine for the first time in the challenge. Our results reveal that 1) the acute locomotor response to amphetamine does not predict vulnerability to behavioral sensitization and 2) differences in vulnerability to sensitization may involve distinctive cellular adaptations at particular brain locations which may be related to addictive vulnerability.

Dysregulation of dynorphins in Alzheimer disease.

The opioid peptides dynorphins may be involved in pathogenesis of Alzheimer disease (AD) by inducing neurodegeneration or cognitive impairment. To test this hypothesis, the dynorphin system was analyzed in postmortem samples from AD and control subjects, and subjects with Parkinson or cerebro-vascular diseases for comparison. Dynorphin A, dynorphin B and related neuropeptide nociceptin were determined in the Brodmann area 7 by radioimmunoassay. The precursor protein prodynorphin, processing convertase PC2 and the neuroendocrine pro7B2 and 7B2 proteins required for PC2 maturation were analyzed by Western blot. AD subjects displayed robustly elevated levels of dynorphin A and no differences in dynorphin B and nociceptin compared to controls. Subjects with Parkinson or cerebro-vascular diseases did not differ from controls with respect to any of the three peptides. PC2 levels were also increased, whereas, those of prodynorphin and pro7B2/7B2 were not changed in AD. Dynorphin A levels correlated with the neuritic plaque density. These results along with the known non-opioid ability of dynorphin A to induce neurodegeneration suggest a role for this neuropeptide in AD neuropathology.

Divergent anatomical pattern of D1 and D3 binding and dopamine- and cyclic AMP-regulated phosphoprotein of 32 kDa mRNA expression in the Roman rat strains: Implications for drug addiction.

Autoradiography analysis of D1, D2 and D3 dopamine receptors and in situ hybridization analysis of mRNA for dopamine and cAMP regulated phosphoprotein of 32 kDa (DARPP-32) were performed in brains of naïve Roman high avoidance (RHA) and Roman low avoidance (RLA) inbred rats. These strains, genetically selected for high (RHA) or extremely low (RLA) active avoidance acquisition in the two-way shuttle box, differ in indices of dopaminergic activity along with sensation/novelty and substance-seeking behavioral profiles. The present study shows no differences in D2 receptor binding between the two strains. In contrast, the D1 and D3 receptor binding in the nucleus accumbens was higher in RHA-I rats, whereas RLA-I rats show higher D3 binding in the Calleja islands. Together with previous evidence showing behavioral and presynaptic differences related to the dopamine system, the present results suggest a higher dopaminergic tone at the nucleus accumbens shell in RHA-I rats. Besides, the comparison of the expression pattern of DARPP-32 mRNA with that of dopamine receptor binding revealed a mismatch in some amygdala nuclei. In some cortical structures (prelimbic and cingulate cortices, the dentate gyrus) as well as in the central amygdala, RHA-I rats showed higher DARPP-32 mRNA expression than RLA-I rats. Hence, RHA-I and RLA-I rats may be a useful tool to identify dopamine-related mechanisms that predispose to drug and alcohol dependence.

Study of molecular events in cells by fluorescence correlation spectroscopy.

To understand processes in a living cell, sophisticated and creative approaches are required that can be used for gathering quantitative information about large number of components interacting across temporal and spatial scales without major disruption of the integral network of processes. A physical method of analysis that can meet these requirements is fluorescence correlation spectroscopy (FCS), which is an ultrasensitive and non-invasive detection method capable of single-molecule and real-time resolution. Since its introduction about 3 decades ago, this until recently emerging technology has reached maturity. As commercially built equipment is now available, FCS is extensively applied for extracting biological information from living cells unattainable by other methods, and new biological concepts are formulated based on findings by FCS. In this review, we focus on examples in the field of molecular cellular biology. The versatility of the technique in this field is illustrated in studies of single-molecule dynamics and conformational flexibility of proteins, and the relevance of conformational flexibility for biological functions regarding the multispecificity of antibodies, modulation of activity of C5a receptors in clathrin-mediated endocytosis and multiplicity of functional responses mediated by the p53 tumor suppressor protein; quantitative characterization of physicochemical properties of the cellular interior; protein trafficking; and ligand-receptor interactions. FCS can also be used to study cell-to-cell communication, here exemplified by clustering of apoptotic cells via bystander killing by hydrogen peroxide.

A systematic genomewide linkage study in 353 sib pairs with schizophrenia.

We undertook a genomewide linkage study in a total of 353 affected sib pairs (ASPs) with schizophrenia. Our sample consisted of 179 ASPs from the United Kingdom, 134 from Sweden, and 40 from the United States. We typed 372 microsatellite markers at approximately 10-cM intervals. Our strongest finding was a LOD score of 3.87 on chromosome 10q25.3-q26.3, with positive results being contributed by all three samples and a LOD-1 interval of 15 cM. This finding achieved genomewide significance (P<.05), on the basis of simulation studies. We also found two regions, 17p11.2-q25.1 (maximum LOD score [MLS] = 3.35) and 22q11 (MLS = 2.29), in which the evidence for linkage was highly suggestive. Linkage to all of these regions has been supported by other studies. Moreover, we found strong evidence for linkage (genomewide P<.02) to 17p11.2-q25.1 in a single pedigree with schizophrenia. In our view, the evidence is now sufficiently compelling to undertake detailed mapping studies of these three regions.

Acquisition, expression, and reinstatement of ethanol-induced conditioned place preference in mice: effects of opioid receptor-like 1 receptor agonists and naloxone.

The ability of the two opioid receptor-like receptor 1 (ORL1) agonists nociceptin (5 nmol i.c.v.) and synthetic (1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one hydrochloride (Ro 64-6198; 0.1, 0.3, and 1.0 mg/kg i.p.) and the opioid antagonist naloxone (0.1, 1.0, and 10.0 mg/kg s.c.) to modify ethanol-induced conditioned place preference was examined in NMRI male mice. The ORL1 agonists were found to significantly reduce the acquisition, expression, and ethanol-induced reinstatement of conditioned place preference. Unlike the ORL1 agonists, naloxone at the doses relevant for opioid receptor blockade failed to significantly influence the acquisition of ethanol-induced conditioned place preference. However, naloxone at 1.0 but not 0.1 mg/kg s.c. potently blocked the expression of ethanol-induced conditioned place preference and significantly inhibited ethanol-induced reinstatement of the conditioned place preference after extinction. Separate experiments indicated that nociceptin and Ro 64-6198 are both devoid of reinforcing or aversive properties. Naloxone, however, at 1.0 and 10.0 mg/kg, produced conditioned place aversion, indicating motivational properties of its own. Both nociceptin and Ro 64-6198 reduced locomotor activity after acute administration. However, tolerance developed very quickly to this effect and already after three i.c.v. (or i.p.) injections, there was no significant reduction of locomotor activity. It is concluded that ORL1 agonists can modulate the acquisition, expression, and reinstatement of the conditioned reinforcing effects of ethanol with no reinforcing or aversive properties of their own. This property might be a potential advantage in the treatment of alcoholism compared with nonselective opioid antagonist naltrexone.

Cytotoxic effects of dynorphins through nonopioid intracellular mechanisms.

Dynorphin A, a prodynorphin-derived peptide, is able to induce neurological dysfunction and neuronal death. To study dynorphin cytotoxicity in vitro, prodynorphin-derived peptides were added into the culture medium of nonneuronal and neuronal cells or delivered into these cells by lipofection or electroporation. Cells were unaffected by extracellular exposure when peptides were added to the medium. In contrast, the number of viable cells was significantly reduced when dynorphin A or "big dynorphin," consisting of dynorphins A and B, was transfected into cells. Big dynorphin was more potent than dynorphin A, whereas dynorphin B; dynorphin B-29; [Arg(11,13)]-dynorphin A(-13)-Gly-NH-(CH(2))(5)-NH(2), a selective kappa-opioid receptor agonist; and poly-l-lysine, a basic peptide more positively charged than big dynorphin, failed to affect cell viability. The opioid antagonist naloxone did not prevent big dynorphin cytotoxicity. Thus, the toxic effects were structure selective but not mediated through opioid receptors. When big dynorphin was delivered into cells by lipofection, it became localized predominantly in the cytoplasm and not in the nuclei. Big dynorphin appeared to induce toxicity through an apoptotic mechanism that may involve synergistic interactions with the p53 tumor-suppressor protein. It is proposed that big dynorphin induces cell death by virtue of its net positive charge and clusters of basic amino acids that mimic (and thereby perhaps interfere with) basic domains involved in protein-protein interactions. These effects may be relevant for a pathophysiological role of dynorphins in the brain and spinal cord and for control of death of tumor cells, which express prodynorphin at high levels.

Oscillation of pain intensity during adenosine infusion. Relationship to beta-endorphin and sympathetic tone.

Adenosine is a neuromodulator with both excitatory and inhibitory effects dependent in part upon preconditions; it can act as an algesic or an analgesic agent. Previously we found variations of pain intensity during constant infusion of adenosine. We therefore quantified pain intensity during constant infusion of adenosine at a rate of 140 microg/kg/min intravenously in healthy volunteers, placebo controlled, double blind, and the relation to hemodynamic, vasomotor and sudomotor responses of the sympathetic nervous system and to the role of peripheral beta-endorphin response. The perceived chest pain during adenosine infusion showed an oscillatory pattern. Painful periods of about 30s were interrupted by painfree periods, and pain was always preceded by an increase in vasomotor sympathetic activity and by increased sudomotor activity. Plasma beta-endorphin values were heterogenous but exhibited an increase during infusion.

p53 Latency. C-terminal domain prevents binding of p53 core to target but not to nonspecific DNA sequences.

The p53 transcription factor is either latent or activated through multi-site phosphorylation and acetylation of the negative regulatory region in its C-terminal domain (CTD). How CTD modifications activate p53 binding to target DNA sequences via its core domain is still unknown. It has been proposed that nonmodified CTD interacts either with the core domain or with DNA preventing binding of the core domain to DNA and that the fragments of the CTD regulatory region activate p53 by interfering with these interactions. We here characterized the sequence and target specificity of p53 activation by CTD fragments, interaction of activating peptides with p53 and target DNA, and interactions of "latent" p53 with DNA by a band shift assay and by fluorescence correlation spectroscopy. In addition to CTD fragments, several long basic peptides activated p53 and also transcription factor YY1. These peptides and CTD aggregated target DNA but apparently did not interact with p53. The potency to aggregate DNA correlated with the ability to activate p53, suggesting that p53 binds to target sequences upon interactions with tightly packed DNA in aggregates. Latent full-length p53 dissociated DNA aggregates via its core and CTD, and this effect was potentiated by GTP. Latent p53 also formed complexes via both its core and CTD with long nontarget DNA molecules. Such p53-DNA interactions may occur if latent p53 binding to DNA via CTD prevents the interaction of the core domain with target DNA sites but not with nonspecific DNA sequences.

Antinociceptive effect produced by intracerebroventricularly administered dynorphin A is potentiated by p-hydroxymercuribenzoate or phosphoramidon in the mouse formalin test.

The antinociceptive effects of intracerebroventricularly (i.c.v.) administered dynorphin A, an endogenous agonist for kappa-opioid receptors, in combination with various protease inhibitors were examined using the mouse formalin test in order to clarify the nature of the proteases involved in the degradation of dynorphin A in the mouse brain. When administered i.c.v. 15 min before the injection of 2% formalin solution into the dorsal surface of a hindpaw, 1-4 nmol dynorphin A produced a dose-dependent reduction of the nociceptive behavioral response consisting of licking and biting of the injected paw during both the first (0-5 min) and second (10-30 min) phases. When co-administered with p-hydroxymercuribenzoate (PHMB), a cysteine protease inhibitor, dynorphin A at the subthreshold dose of 0.5 nmol significantly produced an antinociceptive effect during the second phase. This effect was significantly antagonized by nor-binaltorphimine, a selective kappa-opioid receptor antagonist, but not by naltrindole, a selective delta-opioid receptor antagonist. At the same dose of 0.5 nmol, dynorphin A in combination with phosphoramidon, an endopeptidase 24.11 inhibitor, produced a significant antinociceptive effect during both phases. The antinociceptive effect was significantly antagonized by naltrindole, but not by nor-binaltorphimine. Phenylmethanesulfonyl fluoride (PMSF), a serine protease inhibitor, bestatin, a general aminopeptidase inhibitor, and captopril, an angiotensin-converting enzyme inhibitor, were all inactive. The degradation of dynorphin A by mouse brain extracts in vitro was significantly inhibited only by the cysteine protease inhibitors PHMB and N-ethylmaleimide, but not by PMSF, phosphoramidon, bestatin or captopril. The present results indicate that cysteine proteases as well as endopeptidase 24.11 are involved in two steps in the degradation of dynorphin A in the mouse brain, and that phosphoramidon inhibits the degradation of intermediary delta-opioid receptor active fragments enkephalins which are formed from dynorphin A.

Endomorphin-2 but not Leu-enkephalin modulates spatial learning when microinjected in the CA3 region of the rat hippocampus.

The recently discovered endogenous mu-selective opioid peptide, endomorphin-2, and the endogenous delta-selective opioid peptide, Leu-enkephalin, were tested for their ability to affect spatial learning in the Morris water task. It was found that microinjection of 10 nmol endomorphin-2 into the CA3 region of the rat hippocampus significantly impaired spatial learning. However, the two lower doses tested, 3.3 and 1 nmol, had no effect in this test. Leu-enkephalin did not have any effect on spatial learning at the two doses tested (10 and 3.3 nmol). Neither peptide had any effect on motor performance as measured by swim speed. The results indicate that mu-receptors in the CA3 region of the rat hippocampus are more relevant than delta-receptors for spatial learning.

Dose- and time-dependent bimodal effects of kappa-opioid agonists on locomotor activity in mice.

The kappa-opioid agonists U50488H, bremazocine, and BRL52537, and the mu-opioid agonist morphine were compared in their ability to modify spontaneous motor activity in male NMRI mice. Higher, analgesic doses of the kappa-agonists reduced rearing, motility, and locomotion in nonhabituated mice. These effects, as well as the analgesic action of U50488H, were blocked by the selective kappa-opioid antagonists nor-binaltorphimine and DIPPA. In contrast, lower, subanalgesic doses (1.25 and 2.5 mg/kg for U50488H; 0.15 and 0.075 mg/kg for bremazocine, and 0.1 mg/kg for BRL52537) time dependently increased motor activity. The stimulatory effects of U50488H and bremazocine were not observed in habituated animals and were reduced by dopamine depletion. Surprisingly, the stimulatory effects of U50488H and bremazocine were not blocked by nor-binaltorphimine and DIPPA but they were completely eliminated by naloxone (0.1 mg/kg). The effects of morphine were dose-dependent; an initial limited suppression was followed by increased motility and locomotion (but not rearing) with a peak effect at 20 mg/kg both in habituated and nonhabituated mice. The selective mu-opioid antagonist beta-funaltrexamine blocked morphine-induced motor stimulation and analgesia but failed to affect the analgesic and motor stimulatory effects of U50488H. The results indicate that kappa-opioid agonists interact with different functional subtypes of opioid receptors. A stimulatory, naloxone-sensitive but nor-binaltorphimine- and DIPPA-insensitive subtype of opioid receptor appears to operate only when the dopamine system is tonically active in nonhabituated animals. At higher doses, kappa-agonists produce analgesia and motor suppression, effects mediated by a "classic" (inhibitory) kappa-opioid receptor.

Nociceptin/orphanin FQ metabolism and bioactive metabolites.

The endogenous ligand for the orphan NOR receptor (earlier named ORL1) was recently discovered. This ligand, nociceptin/orphanin FQ is involved in a number of pharmacological actions in the CNS, including modulation of pain and cognition. However, its specific physiological role remains to be determined. Two major pathways of metabolism have been identified; the action of aminopeptidase(s) that prominently occurs in plasma, and endopeptidase activity that successively generates the N-terminal 1-13 and 1-9 fragments. Both pathways result in fragments that are inactive at the NOR receptor. However, short N-terminal fragments appear to be active in blocking the release of substance P from primary afferent C-fiber terminals in the dorsal spinal cord. The same endopeptidase(s) may also be involved in the fragmentation of dynorphin A since the inhibitor profile is similar. Enzyme activity is upregulated by morphine using either peptide as substrate that may lead to pharmacological interactions.

Clustering of apoptotic cells via bystander killing by peroxides.

Clustering of apoptotic cells is a characteristic of many developing or renewing systems, suggesting that apoptotic cells kill bystanders. Bystander killing can be triggered experimentally by inducing apoptosis in single cells and may be based on the exchange of as yet unidentified chemical cell death signals between nearby cells without the need for cell-to-cell communication via gap junctions. Here we demonstrate that apoptotic cell clusters occurred spontaneously, after serum deprivation or p53 transfection in cell monolayers in vitro. Clustering was apparently induced through bystander killing by primary apoptotic cells. Catalase, a peroxide scavenger, suppressed bystander killing, suggesting that hydrogen peroxide generated by apoptotic cells is the death signal. Although p53 expression increased the number of apoptoses, clustering was found to be similar around apoptotic cells whether or not p53 was expressed, indicating that there is no specific p53 contribution to bystander killing. Bystander killing through peroxides emitted by apoptotic cells may propagate tissue injury in different pathological situations and be relevant in chemo-, gamma-ray, and gene therapy of cancer.

From opiate pharmacology to opioid peptide physiology.

This is a personal account of how studies of the pharmacology of opiates led to the discovery of a family of endogenous opioid peptides, also called endorphins. The unique pharmacological activity profile of opiates has an endogenous counterpart in the enkephalins and beta-endorphin, peptides which also are powerful analgesics and euphorigenic agents. The enkephalins not only act on the classic morphine (mu-) receptor but also on the delta-receptor, which often co-exists with mu-receptors and mediates pain relief. Other members of the opioid peptide family are the dynorphins, acting on the kappa-receptor earlier defined as precipitating unpleasant central nervous system (CNS) side effects in screening for opiate activity, A related peptide, nociceptin is not an opioid and acts on the separate NOR-receptor. Both dynorphins and nociceptin have modulatory effects on several CNS functions, including memory acquisition, stress and movement. In conclusion, a natural product, morphine and a large number of synthetic organic molecules, useful as drugs, have been found to probe a previously unknown physiologic system. This is a unique development not only in the neuropeptide field, but in physiology in general.