Prenatal one-carbon metabolism dysregulation programs schizophrenia-like deficits.

The methionine-folate cycle-dependent one-carbon metabolism is implicated in the pathophysiology of schizophrenia. Since schizophrenia is a developmental disorder, we examined the effects that perturbation of the one-carbon metabolism during gestation has on mice progeny. Pregnant mice were administered methionine equivalent to double their daily intake during the last week of gestation. Their progeny (MET mice) exhibited schizophrenia-like social deficits, cognitive impairments and elevated stereotypy, decreased neurogenesis and synaptic plasticity, and abnormally reduced local excitatory synaptic connections in CA1 neurons. Neural transcript expression of only one gene, encoding the Npas4 transcription factor, was >twofold altered (downregulated) in MET mice; strikingly, similar Npas4 downregulation occurred in the prefrontal cortex of human patients with schizophrenia. Finally, therapeutic actions of typical (haloperidol) and atypical (clozapine) antipsychotics in MET mice mimicked effects in human schizophrenia patients. Our data support the validity of MET mice as a model for schizophrenia, and uncover methionine metabolism as a potential preventive and/or therapeutic target.

The melanin-concentrating hormone (MCH) system in an animal model of depression-like behavior.

Selective breeding for divergence in locomotion in a novel environment (bHR, bred High-Responder; bLR, bred Low-Responder) correlates with stress-reactivity, spontaneous anxiety-like behaviors and predicts vulnerability in a rodent model of depression. Identifying genetic factors that may account for such vulnerability are key determinants not only for the illness outcome but also for the development of better-tailored treatment options. Melanin-concentrating hormone (MCH) is a neuropeptide that exhibits some of the hallmarks of a regulator of affective states. The aim of this study was to ascertain the role of the MCH system in depression-like behaviors in bHR vs. bLR rats. bLR rats showed a 44% increase in hypothalamic pMCH mRNA and a 14% decrease in hippocampal CA1 MCH1R mRNA when compared to bHR rats. Interestingly, the amount of time that rats spent immobile in the FST (depressive-like behavior) correlated positively with the amount of hypothalamic pMCH mRNA and negatively with that of hippocampal CA1 MCH1R. The results indicate that the bLR-bHR is a useful rat model to investigate individual basal genetic differences that participate in the monitoring of emotional responsiveness (i.e., depression- and anxiety-like behaviors). They also point to the MCH system (i.e., chronically higher pMCH expression and consequently receptor down-regulation) as a candidate biomarker for the severity of depressive-like behavior. The data indicate that MCH1R participates in the modulation of depression-like behavior through a process that involves the CA1 region of the hippocampus, supporting the possible use of MCH1R antagonists in the treatment of depression.

Orphan GPCR research.

Orphan G protein-coupled receptors (GPCRs) are receptors lacking endogenous ligands. Found by molecular biological analyses, they became the roots of reverse pharmacology, in which receptors are attempted to be matched to potential transmitters. Later, when high-throughput screening technology was applied to reverse pharmacology, dozens of orphan GPCRs became deorphanized. Furthermore, novel neuropeptides were discovered. This review retraces the history of the orphan GPCRs and of the discoveries of their endogenous ligands, it also discusses the difficulties that the search for new ligands is presently encountering.

Fusion of diphtheria toxin and urotensin II produces a neurotoxin selective for cholinergic neurons in the rat mesopontine tegmentum.

Urotensin II is a neuropeptide first isolated from fish and later found in mammals: where it has potent cardiovascular, endocrine and behavioral effects. In rat brain the urotensin II receptor (UII-R) is predominately expressed in the cholinergic neurons of the pedunculopontine (PPTg) and laterodorsal tegmental nuclei. Typically, the function of the PPTg has been examined using excitotoxins, destroying both cholinergic and non-cholinergic neurons, which confounds interpretation. We took advantage of UII-R's unique expression profile, by combining UII with diphtheria toxin, to engineer a toxin specific for cholinergic neurons of the PPTg. In vitro, two different toxin constructs were shown to selectively activate UII-R (average EC50 approximately 30 nmol/L; calcium mobility assay) and to be 10,000-fold more toxic to UII-R expressing CHO cells, than wildtype cells (average LD50 approximately 2 nmol/L; cell viability). In vivo, pressure injection into the PPTg of rats, resulted in specific loss of choline transporter and NADPH diaphorase positive neurons known to express the UII-R. The lesions developed over time, resulting in the loss of over 80% of cholinergic neurons at 21 days, with little damage to surrounding neurons. This is the first highly selective molecular tool for the depletion of mesopontine cholinergic neurons. The toxin will help to functionally dissect the pedunculopontine and laterodorsal tegmental nuclei, and advance the understanding of the functions of these structures.

The orphanin FQ/nociceptin knockout mouse: a behavioral model for stress responses.

Transgenic mice lacking expression of the OFQ/N precursor protein have provided exciting insights in the physiological functions of this neuropeptide system. While injection of OFQ/N or selective synthetic agonists produces anxiolytic effects in rodents, OFQ/N knockout mice display increased anxiety and impaired adaptation to repeated stress. On the other hand, mice lacking the cognate OFQ/N receptor, ORL1, show improved spatial attention and memory but appear to have normal anxiety and stress behavior. Availability of a selective small molecule OFQ/N antagonist might help clarify this discrepancy.

Prolactin-releasing peptide (PrRP) promotes awakening and suppresses absence seizures.

Prolactin releasing peptide (PrRP) is a recently identified neuropeptide that stimulates prolactin release from pituitary cells. The presence of its receptor outside the hypothalamic-pituitary axis suggests that it may have other functions. We present here evidence that PrRP can modulate the activity of the reticular thalamic nucleus, a brain region with prominent PrRP receptor expression that is critical for sleep regulation and the formation of non-convulsive absence seizures. Intracerebroventricular injection of PrRP (1-10 nmol) into sleeping animals significantly suppresses sleep oscillations and promotes rapid and prolonged awakening. Higher concentrations of PrRP (10-100 nmol) similarly suppress spike wave discharges seen during absence seizures in genetic absence epilepsy rats from Strasbourg, an animal model for this disorder. In concordance with these findings, PrRP suppressed evoked oscillatory burst activity in reticular thalamic slices in vitro. These results indicate that PrRP modulates reticular thalamic function and that activation of its receptor provides a new target for therapies directed at sleep disorders and absence seizures.

The urotensin II receptor is expressed in the cholinergic mesopontine tegmentum of the rat.

Urotensin II (UII) is a peptide known to be a potent vasoconstrictor. The urotensin II receptor (UII-R) is expressed not only in peripheral tissues but also in the brain of rodents. As a basis for studies of UII central nervous system actions, UII-R localization in the rat brain was analyzed by in situ hybridization and by in situ binding. UII-R mRNA was found in the mesopontine tegmental area colocalizing with choline acetyltransferase. Binding sites were detected throughout the brain with the highest levels found in the pedunculopontine tegmental area, the lateral dorsal tegmental area, and the lateral septal, medial habenular, and interpeduncular nuclei. The majority of these brain nuclei are sites of axonal termination originating from the mesopontine areas, suggesting that UII-R is a presynaptic receptor. This distribution of UII-R in the cholinergic mesopontine area indicates that the UII system may be involved in sensory-motor integration and perhaps in central nervous system blood flow.

Endogenous melanin-concentrating hormone receptor SLC-1 in human melanoma SK-MEL-37 cells.

Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide that regulates several physiological functions. The orphan G protein-coupled receptors SLC-1 and MCHR2 were recently found to bind MCH with high affinity. We show here that the human melanoma cell line SK-MEL-37 expresses SLC-1 mRNA but not MCHR2 by RT-PCR analysis and immunofluorescence studies. Using Chinese hamster ovary cells and 293 cells overexpressing SLC-1 by cDNA transfection, it was shown that SLC-1 coupled to both G alpha(i)/G alpha(o) and G alpha(q) proteins. In SK-MEL-37 cells, MCH inhibited forskolin-stimulated cyclic AMP accumulation and induced mitogen-activated protein kinase (MAPK) in a pertussis toxin-(PTX)-sensitive manner. The MAPK activity leads to the production of phosphorylated forms of p42/p44 MAPK. However, an increase in the intracellular free Ca(2+) concentration was not elicited by MCH in SK-MEL-37 cells. These results show that SLC-1 is coupled only to PTX-sensitive G alpha(i)/G alpha(o) in SK-MEL-37 cells. This study provides for the first time a skin-derived cellular model to analyze the molecular mechanism of the MCH signaling pathway.

The carboxyl terminus of the prolactin-releasing peptide receptor interacts with PDZ domain proteins involved in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor clustering.

PDZ domain proteins use the PDZ domain binding motif to bind to the C-terminal sequence of membrane proteins to help scaffold them and spatially organize the components of the intracellular signaling machinery. We have identified a sequence at the C terminus of a G protein-coupled receptor, the PrRP receptor, that shares similarities with the C-terminal sequence of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPA-R) subunits that interact with PDZ domain proteins. When coexpressed in human embryonic kidney 293 cells, PrRP receptor was able to coimmunoprecipitate the three PDZ domain proteins known to interact with AMPA receptors: glutamate receptor interacting protein (GRIP), AMPA binding protein (ABP), and protein that interacts with C-kinase (PICK1), but not the PDZ domain protein PSD-95, which does not interact with AMPA receptors. These interactions are sequence-selective as determined by mutagenesis. Furthermore, we show that PrRP receptor forms intracellular clusters when coexpressed with PICK1, and that this clustering effect is dependent on the interaction between the PICK1 PDZ domain and the last four amino acids of PrRP receptor. We found that PrRP receptor interaction with GRIP is not protein kinase C-regulated but may be regulated by other unidentified kinase because okadaic acid dramatically reduced GRIP interaction. By in situ hybridization, we show that the PrRP receptor is expressed in neurons that also express these PDZ domain proteins. We thus demonstrate that PrRP receptor interacts with the same PDZ domain proteins as the AMPA-Rs, raising the possibility that these two proteins could be scaffolded together at the synapse. These results may help to gain important insights into PrRP functions within the central nervous system.

Expression of the melanin-concentrating hormone (MCH) receptor mRNA in the rat brain.

The melanin-concentrating hormone (MCH) system is thought to be an important regulator of food intake. Recently the orphan G protein-coupled receptor SLC-1 was identified as the MCH receptor (MCHR). Preliminary analyses of MCHR mRNA distribution have supported a role for the MCH system in nutritional homeostasis. We report here a complete anatomical distribution of the MCHR mRNA. We have found high levels of expression of MCHR mRNA in most anatomical areas implicated in control of olfaction, with the exception of the main olfactory bulb. Dense labeling was also detected in the hippocampal formation, subiculum, and basolateral amygdala, all of which are important in learning and memory, and in the shell of the nucleus accumbens, a substrate for motivated behavior and feeding. Within the hypothalamus, MCHR mRNA was moderately expressed in the ventromedial nucleus, arcuate nucleus, and zona incerta, all of which serve key roles in the neuronal circuitry of feeding. In the brainstem, strong expression was observed in the locus coeruleus, which is implicated in arousal, as well as in nuclei that contribute to orofacial function and mastication, including the facial, hypoglossal, motor trigeminal, and dorsal motor vagus nuclei. In most regions there was a good correspondence between MCHR mRNA distribution and that of MCH-immunoreactive fibers. Taken together, these data suggest that MCH may act at various levels of the brain to integrate various aspects of feeding behavior. However, the extensive MCHR distribution throughout the brain suggests that this receptor may play a role in other functions, most notably reinforcement, arousal, sensorimotor integration, and autonomic control.

Novel neurotransmitters as natural ligands of orphan G-protein-coupled receptors.

The "orphan" G-protein-coupled receptors (GPCRs) are cloned GPCRs that bind unknown ligands. Since 1995, nineteen orphan GPCRs have been used as targets to identify and isolate their natural ligands via the application of the "orphan receptor strategy". These ligands are peptides, lipids or biogenic amines, and act as transmitter molecules. One nucleotide-sugar derivative and six peptides or peptide families identified through this strategy are novel and have already enriched our understanding of various brain functions.

Molecular cloning and characterization of a second human cysteinyl leukotriene receptor: discovery of a subtype selective agonist.

The cysteinyl leukotrienes (CysLTs) are potent biological mediators in the pathophysiology of inflammatory diseases, in particular of airway obstruction in asthma. Pharmacological studies have suggested the existence of at least two types of CysLT receptors, designated CysLT(1) and CysLT(2). The CysLT(1) receptor has been cloned recently. Here we report the molecular cloning, expression, localization, and functional characterization of a human G protein-coupled receptor that has the expected characteristics of a CysLT(2) receptor. This new receptor is selectively activated by nanomolar concentrations of CysLTs with a rank order potency of LTC(4) = LTD(4) >> LTE(4). The leukotriene analog BAY u9773, reported to be a dual CysLT(1)/CysLT(2) antagonist, was found to be an antagonist at CysLT(1) sites but acted as a partial agonist at this new receptor. The structurally different CysLT(1) receptor-selective antagonists zafirlukast, montelukast, and MK-571 did not inhibit the agonist-mediated calcium mobilization of CysLT(2) receptors at physiological concentrations. Localization studies indicate highest expression of CysLT(2) receptors in adrenal glands, heart, and placenta; moderate levels in spleen, peripheral blood leukocytes, and lymph nodes; and low levels in the central nervous system and pituitary. The human CysLT(2) receptor gene is located on chromosome 13q14.12-21.1. The new receptor exhibits all characteristics of the thus far poorly defined CysLT(2) receptor. Moreover, we have identified BAY u9773 as a CysLT(2) selective agonist, which could prove to be of immediate use in understanding the functional roles of the CysLT(2) receptor.

Melanin-concentrating hormone receptor: an orphan receptor fits the key.

The melanin-concentrating hormone (MCH), a hypothalamic peptide, was identified initially in teleost fish as a regulator of pigmentary changes in background adaptation, and was later also found, in mammals, to be a regulator of feeding and energy homeostasis. Its specific receptor remained an enigma until very recently when it was identified as the orphan G-protein-coupled receptor SLC-1. This review focuses on the identification, structure and signaling of the MCH receptor and discusses some of the implications of its discovery.

The orphanin FQ/nociceptin gene: structure, tissue distribution of expression and functional implications obtained from knockout mice.

Like other neuropeptides, orphanin FQ/nociceptin (OFQ/N) is encoded by a larger precursor protein. The cDNA for the OFQ/N precursor has been cloned from human, rat, mouse and bovine tissue demonstrating that this peptidergic system serves important functions that have been conserved during evolution. The structural organization of the precursor protein is similar to opioid peptide precursors, supporting the view of a common origin for the opioid systems and the OFQ/N system. In addition to OFQ/N, the precursor may encode two other biologically active peptides. Anatomic studies have revealed high levels of expression of the OFQ/N messenger RNA in brain structures involved in sensory, emotional and cognitive processing. In particular, high levels of OFQ/N mRNA were detected in the limbic system, underlining the stress attenuating activities that have been described as an important function of OFQ/N. Recently, mutant mice have been generated that lack the precursor protein of OFQ/N to further define the physiological functions of the OFQ/N system. The OFQ/N-deficient mice are characterized by an increased sensitivity to stressful stimuli and a lack of habituation to chronic and repeated stress. This review will summarize recent findings on the molecular biology of the OFQ/N precursor and relate it to possible physiological functions of this newly discovered neuropeptide system.

Orphan receptors, novel neuropeptides and reverse pharmaceutical research.

By the beginning of the next millennium, the search for the natural ligands of the orphan G-protein-coupled receptors will lead to the discovery of so many new neuropeptides that it may well double their present number. This bounty of new tools will direct us to new insights in brain function and to better understanding of brain disorders. It is expected that the novel neuropeptides will have a particular impact on molecular psychiatry. In view of their potential, the novel neuropeptides should also become the focus of drug discovery programs. It is hoped that these programs will be initiated at an early stage, when understanding of novel neuropeptide function has not necessarily been reached, to allow for the design of neuropeptide chemical surrogates that are crucial to the study of the novel neuropeptide system and may serendipitously develop into highly successful drugs.

Targeted disruption of the orphanin FQ/nociceptin gene increases stress susceptibility and impairs stress adaptation in mice.

The neuropeptide orphanin FQ (also known as nociceptin; OFQ/N) has been implicated in modulating stress-related behavior. OFQ/N was demonstrated to reverse stress-induced analgesia and possess anxiolytic-like activity after central administration. To further study physiological functions of OFQ/N, we have generated OFQ/N-deficient mice by targeted disruption of the OFQ/N gene. Homozygous mice display increased anxiety-like behavior when exposed to a novel and threatening environment. OFQ/N-null mice show elevated basal pain threshold but develop normal stress-induced analgesia. Interestingly, these mice show impaired adaptation to repeated stress when compared with wild-type mice, whereas their performance in spatial learning remained unaffected. Basal and poststress plasma corticosterone levels were found to be elevated in OFQ/N-deficient animals. Thus, OFQ/N appears to be crucially involved in the neurobiological regulation of stress-coping behavior and fear.

Orphan receptors and the concept of reverse physiology: discovery of the novel neuropeptide orphanin FQ/nociceptin.

The cloning of numerous orphan members from the supergene family of G protein-coupled receptors implies the existence of many as yet undiscovered neurotransmitters and neuropeptides. Recently, new technologies were developed to isolate natural ligands for orphan receptors, using the receptor as a biological sensor during the purification process. This manuscript will present the concept and technology of an approach which starts from a cloned receptor to ultimately describe the physiological functions of the transmitter system. This strategy inverts the classical order of biomedical research and was thus termed "reverse physiology". The first natural ligand isolated by this strategy is a peptide with significant similarity to the opioid peptides and has been named orphanin FQ or nociceptin (OFQ/NOC). Evidence for characterizing OFQ/NOC as a genuine neuropeptide will be reviewed. OFQ/NOC is biosynthetically derived from a larger precursor protein which may encode additional bioactive peptides. Since its discovery, a large number of studies have described numerous physiological functions of OFQ/NOC. Because of its relation to the opioid system, much attention has been focused on the involvement of OFQ/NOC in nociception, sometimes with controversial results. However, the pharmacological profile of the OFQ/NOC system suggests a clear separation from the opioids. The discovery of OFQ/NOC and the subsequent analyses of its physiological functions is an example which has already been followed by the identification of two other novel neuropeptides. The orphan receptor strategy holds a lot of promises for the postgenomic era, helping to fill the vast amount of sequence data with life.

Opioid receptor-like (ORL1) receptor distribution in the rat central nervous system: comparison of ORL1 receptor mRNA expression with (125)I-[(14)Tyr]-orphanin FQ binding.

The recently discovered neuropeptide orphanin FQ (OFQ), and its opioid receptor-like (ORL1) receptor, exhibit structural features suggestive of the micro, kappa, and delta opioid systems. The anatomic distribution of OFQ immunoreactivity and mRNA expression has been reported recently. In the present analysis, we compare the distribution of orphanin receptor mRNA expression with that of orphanin FQ binding at the ORL1 receptor in the adult rat central nervous system (CNS). By using in vitro receptor autoradiography with (125)I-[(14)Tyr]-OFQ as the radioligand, orphanin receptor binding was analyzed throughout the rat CNS. Orphanin binding sites were densest in several cortical regions, the anterior olfactory nucleus, lateral septum, ventral forebrain, several hypothalamic nuclei, hippocampal formation, basolateral and medial amygdala, central gray, pontine nuclei, interpeduncular nucleus, substantia nigra, raphe complex, locus coeruleus, vestibular nuclear complex, and the spinal cord. By using in situ hybridization, cells expressing ORL1 mRNA were most numerous throughout multiple cortical regions, the anterior olfactory nucleus, lateral septum, endopiriform nucleus, ventral forebrain, multiple hypothalamic nuclei, nucleus of the lateral olfactory tract, medial amygdala, hippocampal formation, substantia nigra, ventral tegmental area, central gray, raphe complex, locus coeruleus, multiple brainstem motor nuclei, inferior olive, deep cerebellar nuclei, vestibular nuclear complex, nucleus of the solitary tract, reticular formation, dorsal root ganglia, and spinal cord. The diffuse distribution of ORL1 mRNA and binding supports an extensive role for orphanin FQ in a multitude of CNS functions, including motor and balance control, reinforcement and reward, nociception, the stress response, sexual behavior, aggression, and autonomic control of physiologic processes.

Molecular characterization of the melanin-concentrating-hormone receptor.

Orphan G-protein-coupled receptors (GPCRs) are cloned proteins with structural characteristics common to the GPCRs but that bind unidentified ligands. Orphan GPCRs have been used as targets to identify novel transmitter molecules. Here we describe the isolation from brain extracts and the characterization of the natural ligand of a particular orphan GPCR (SLC-1) that is sequentially homologous to the somatostatin receptors. We show that the natural ligand of this receptor is the neuropeptide melanin-concentrating hormone (MCH). MCH is a cyclic peptide that regulates a variety of functions in the mammalian brain, in particular feeding behaviour. We demonstrate that nanomolar concentrations of MCH strongly activate SLC-1-related pathways through G(alpha)i and/or G(alpha)q proteins. We have analysed the tissue localization of the MCH receptor and find that it is expressed in several brain regions, in particular those involved in olfactory learning and reinforcement mechanisms, indicating that therapies targeting the MCH receptor should act on the neuronal regulation of food consumption.