Roman Artymyshyn

Neurogenesis-Dependent and -Independent Effects of Fluoxetine in an Animal Model of Anxiety/Depression

Understanding the physiopathology of affective disorders and their treatment relies on the availability of experimental models that accurately mimic aspects of the disease. Here we describe a mouse model of an anxiety/depressive-like state induced by chronic corticosterone treatment. Furthermore, chronic antidepressant treatment reversed the behavioral dysfunctions and the inhibition of hippocampal neurogenesis induced by corticosterone treatment. In corticosterone-treated mice where hippocampal neurogenesis is abolished by X-irradiation, the efficacy of fluoxetine is blocked in some, but not all, behavioral paradigms, suggesting both neurogenesis-dependent and -independent mechanisms of antidepressant action. Finally, we identified a number of candidate genes, the expression of which is decreased by chronic corticosterone and normalized by chronic fluoxetine treatment selectively in the hypothalamus. Importantly, mice deficient in one of these genes, beta-arrestin 2, displayed a reduced response to fluoxetine in multiple tasks, suggesting that beta-arrestin signaling is necessary for the antidepressant effects of fluoxetine.

Trace amines: Identification of a family of mammalian G protein-coupled receptors

Tyramine, β-phenylethylamine, tryptamine, and octopamine are biogenic amines present in trace levels in mammalian nervous systems. Although some “trace amines” have clearly defined roles as neurotransmitters in invertebrates, the extent to which they function as true neurotransmitters in vertebrates has remained speculative. Using a degenerate PCR approach, we have identified 15 G protein-coupled receptors (GPCR) from human and rodent tissues. Together with the orphan receptor PNR, these receptors form a subfamily of rhodopsin GPCRs distinct from, but related to the classical biogenic amine receptors. We have demonstrated that two of these receptors bind and/or are activated by trace amines. The cloning of mammalian GPCRs for trace amines supports a role for trace amines as neurotransmitters in vertebrates. Three of the four human receptors from this family are present in the amygdala, possibly linking trace amine receptors to affective disorders. The identification of this family of receptors should rekindle the investigation of the roles of trace amines in mammalian nervous systems and may potentially lead to the development of novel therapeutics for a variety of indications.

Identification and Characterization of Two G Protein-coupled Receptors for Neuropeptide FF

The central nervous system octapeptide, neuropeptide FF (NPFF), is believed to play a role in pain modulation and opiate tolerance. Two G protein-coupled receptors, NPFF1 and NPFF2, were isolated from human and rat central nervous system tissues. NPFF specifically bound to NPFF1 (K d = 1.13 nm) and NPFF2 (K d = 0.37 nm), and both receptors were activated by NPFF in a variety of heterologous expression systems. The localization of mRNA and binding sites of these receptors in the dorsal horn of the spinal cord, the lateral hypothalamus, the spinal trigeminal nuclei, and the thalamic nuclei supports a role for NPFF in pain modulation. Among the receptors with the highest amino acid sequence homology to NPFF1 and NPFF2 are members of the orexin, NPY, and cholecystokinin families, which have been implicated in feeding. These similarities together with the finding that BIBP3226, an anorexigenic Y1 receptor ligand, also binds to NPFF1 suggest a potential role for NPFF1 in feeding. The identification of NPFF1 and NPFF2 will help delineate their roles in these and other physiological functions.

CLONED HUMAN AND RAT GALANIN GALR3 RECEPTORS: PHARMACOLOGY AND ACTIVATION OF G-PROTEIN INWARDLY RECTIFYING K+ CHANNELS

The neuropeptide galanin has been implicated in the regulation of processes such as nociception, cognition, feeding behavior, and hormone secretion. Multiple galanin receptors are predicted to mediate its effects, but only two functionally coupled receptors have been reported. We now report the cloning of a third galanin receptor distinct from GALR1 and GALR2. The receptor, termed GALR3, was isolated from a rat hypothalamus cDNA library by both expression and homology cloning approaches. The rat GALR3 receptor cDNA can encode a protein of 370 amino acids with 35% and 52% identity to GALR1 and GALR2, respectively. Localization of mRNA by solution hybridization/RNase protection demonstrates that the GALR3 transcript is widely distributed, but expressed at low abundance, with the highest levels in the hypothalamus and pituitary. We also isolated the gene encoding the human homologue of GALR3. The human GALR3 receptor is 90% identical to rat GALR3 and contains 368 amino acids. Binding of porcine 125I-galanin to stably expressed rat and human GALR3 receptors is saturable (rat K D = 0.98 nm and human K D = 2.23 nm) and displaceable by galanin peptides and analogues in the following rank order: rat galanin, porcine galanin ≃ M32, M35 ≃ porcine galanin-(−7 to +29), galantide, human galanin > M40, galanin-(1–16) > [d-Trp2]galanin-(1–29), galanin-(3–29). This profile resembles that of the rat GALR1 and GALR2 receptors with the notable exception that human galanin, galanin-(1–16), and M40 show lower affinity at GALR3. InXenopus oocytes, activation of rat and human GALR3 receptors co-expressed with potassium channel subunits GIRK1 and GIRK4 resulted in inward K+ currents characteristic of Gi/Go-coupled receptors. These data confirm the functional efficacy of GALR3 receptors and further suggest that GALR3 signaling pathways resemble those of GALR1 in that both can activate potassium channels linked to the regulation of neurotransmitter release.

Identification and Characterization of Two Neuromedin U Receptors Differentially Expressed in Peripheral Tissues and the Central Nervous System

Two structurally related, G-protein-coupled receptors were identified as receptors for the neuropeptide, neuromedin U. This peptide is found in highest levels in the gut and genitourinary system where it potently contracts smooth muscle but is also expressed in the spinal cord and discrete regions of the brain. Binding sites for neuromedin U have been characterized in rat uterus, however, little is known about the activity of this peptide in the regions of the central nervous system where it is expressed. The receptors characterized in this report are activated by neuromedin U at nanomolar potency in heterologous expression systems and bind radiolabeled neuromedin U with high affinity. Localization of the receptor RNA by quantitative reverse transcription-polymerase chain reaction in a variety of human tissues shows distinct expression patterns for the two receptors. NMU1 is expressed predominantly in peripheral tissues, whereas NMU2 is more highly expressed in the central nervous system. Identification of neuromedin U receptor subtypes will greatly aid in the determination of the physiological roles of this peptide.

Small-molecule antagonists of melanopsin-mediated phototransduction

Melanopsin, expressed in a subset of retinal ganglion cells, mediates behavioral adaptation to ambient light and other non-image forming photic responses. This has raised the possibility that pharmacological manipulation of melanopsin can modulate several CNS responses including photophobia, sleep, circadian rhythms and neuroendocrine function. Here we describe the identification of a potent synthetic melanopsin antagonist with in vivo activity. Novel sulfonamide compounds inhibiting melanopsin (opsinamides) compete with retinal binding to melanopsin and inhibit its function without affecting rod/cone mediated responses. In vivo administration of opsinamides to mice specifically and reversibly modified melanopsin-dependent light responses including the pupillary light reflex and light aversion. The discovery of opsinamides raises the prospect of therapeutic control of the melanopsin phototransduction system to regulate light-dependent behavior and remediate pathological conditions.

Perspectives for an Integrated Biomarker Approach to Drug Discovery and Development

Today’s psychopharmacological drugs remain focused on targets that were identified serendipitously more than half a century ago. As these targets have not proven to be at the core of the pathophysiology of the major psychiatric disorders, a better understanding of the disease biology seems a crucial step to identify more efficacious treatments. The tools to realize this goal include neuroendocrine, protein, transcription and genetic markers, neuroimaging and neurophysiological approaches. Obviously, the benefit for psychiatric patients of identifying a pattern of blood-based markers that combine information on the disease biology and treatment response would be enormous. Our transcription data from human blood cells suggest that this is a realistic possibility for the future. Ideally, markers identified in patients would be translated into distinct, hypothesis-driven animal models to facilitate conclusions on the potential therapeutic utility of novel compounds. The combined use of disease state and mechanistic models may characterize cellular and molecular mechanisms of various aspects of psychiatric disorders. This information, in turn, could help establish in vitro models that link cellular targets to (novel) pharmacological approaches. With the discussions around DSM-V, it can be hoped that ambitious research agenda will guide and stimulate systematic research into the biology and biological markers of psychiatric disorders.

A classifier driven approach to find biomarkers for affective disorders from transcription profiles in blood

Gene expression profiles in blood are increasingly being used to identify biomarkers for different affective disorders. We have selected a set of 29 genes to generate expression profiles for healthy control subjects as well as for patients diagnosed with acute post-traumatic stress disorder (PTSD) and with borderline personality disorder (BPD). Measurements were performed by quantitative polymerase chain reaction (qPCR). Using the actual data in an anonym-ous form we constructed a series of artificial data sets with known gene expression profiles. These sets were used to test 14 classification algorithms and feature selection methods for their ability to identify the correct expression patterns. Application of the three most effective algorithms to the actual expression data showed that control subjects can be dis-tinguished from BPD patients based on differential expression levels of the gene transcripts Gi2, GR and MAPK14, targets that may have links to stress related diseases. Controls can also be distinguished from acute PTSD patients by differential expression levels of the transcripts for ERK2 and RGS2 that are known to be associated with mood disord-ers and social anxiety. We conclude that it is possible to identify informative transcription profiles in blood samples from individuals with affective disorders.