Joseph A. Tamm

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.

Expression Cloning of a Rat Hypothalamic Galanin Receptor Coupled to Phosphoinositide Turnover

The neuropeptide galanin is widely distributed throughout the central and peripheral nervous systems and participates in the regulation of processes such as nociception, cognition, feeding behavior, and insulin secretion. Multiple galanin receptors are predicted to underlie its physiological effects. We now report the isolation by expression cloning of a rat galanin receptor cDNA distinct from GALR1. The receptor, termed GALR2, was isolated from a rat hypothalamus cDNA library using a125I-porcine galanin (125I-pGAL) binding assay. The GALR2 cDNA encoded a protein of 372 amino acids exhibiting 38% amino acid identity with rat GALR1. Binding of125I-pGAL to transiently expressed GALR2 receptors was saturable (K D = 0.15 nm) and displaceable by galanin peptides and analogues in rank order: porcine galanin ≃ M32 ≃ M35 ≃ M40 ≥ galanin-(1–16) ≃ M15 ≃ [d-Trp2]galanin-(1–29) > C7 ≫ galanin-(3–29). This profile resembles that of the rat GALR1 receptor with the notable exception that [d-Trp2]galanin exhibited significant selectivity for GALR2 over GALR1. Activation of GALR2 receptors with porcine galanin and other galanin analogues increased inositol phospholipid turnover and intracellular calcium levels in stably transfected Chinese hamster ovary cells and generated calcium-activated chloride currents in Xenopus oocytes, suggesting that the rat GALR2 receptor is primarily coupled to the activation of phospholipase C.

Reversal of age-associated cognitive deficits is accompanied by increased plasticity-related gene expression after chronic antidepressant administration in middle-aged mice.

Cognitive decline occurs during healthy aging, even in middle-aged subjects, via mechanisms that could include reduced stem cell proliferation, changed growth factor expression and/or reduced expression of synaptic plasticity genes. Although antidepressants alter these mechanisms in young rodents, their effects in older animals are unclear. In middle-aged mice, we examined the effects of a selective serotonin reuptake inhibitor (fluoxetine) and a multimodal antidepressant (vortioxetine) on cognitive and affective behaviors, brain stem cell proliferation, growth factor and gene expression. Twelve-month-old female C57BL/6 mice exhibited impaired visuospatial memory in the novel object placement (location) task associated with reduced expression of several plasticity-related genes. Chronic treatment with vortioxetine, but not fluoxetine, improved visuospatial memory and reduced depression-like behavior in the forced swim test in middle-aged mice. Vortioxetine, but not fluoxetine, increased hippocampal expression of several neuroplasticity-related genes in middle-aged mice (e.g., Nfkb1, Fos, Fmr1, Camk2a, Arc, Shank1, Nlgn2, and Rab3a). Neither drug reversed the age-associated decrease in stem cell proliferation. Hippocampal growth factor levels were not consistent with behavioral outcomes. Thus, a change in the expression of multiple genes involved in neuronal plasticity by antidepressant treatment was associated with improved cognitive function and a reduction in depression-like behavior in middle-aged mice.

Signal Transduction by GABAB Receptor Heterodimers

GABAB receptors are G-protein-coupled receptors that mediate inhibition throughout the central and peripheral nervous systems. A single cloned receptor, GABABR1, which has at least three alternatively spliced forms, appears to account for the vast majority of binding sites in the brain for high-affinity antagonists. In heterologous expression systems GABABR1 is poorly expressed on the plasma membrane and largely fails to couple to ion channels. A second gene, GABABR2, which exhibits moderately low homology to GABABR1, permits surface expression of GABABR1 and the appearance of baclofen-sensitive K+ and Ca+1 currents. We review the data that supports a model of the native GABAB receptor as a heterodimer composed of GABABR1 and GABABR2 proteins. New data from mutagenesis experiments are presented that point to amino acid residues on GABABR1 critical for ligand activation of the heterodimer. The possible role of GABABR2 in signal transduction is also discussed. The interdependent nature of the two subunits for receptor function makes the GABAB receptor a useful model to explore the larger significance of GPCR dimerization for G-protein activation.

Chronic vortioxetine treatment in rodents modulates gene expression of neurodevelopmental and plasticity markers

The multimodal antidepressant vortioxetine displays an antidepressant profile distinct from those of conventional selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs) and possesses cognitive-enhancing properties in preclinical and clinical studies. Recent studies have begun to investigate molecular mechanisms that may differentiate vortioxetine from other antidepressants. Acute studies in adult rats and chronic studies in a middle-aged mouse model reveal upregulation of several markers that play a central role in synaptic plasticity. However, the effect of chronic vortioxetine treatment on expression of neuroplasticity and neurodevelopmental biomarkers in naïve rats has not been evaluated. In the present study, we demonstrate that vortioxetine at a range of doses regulates expression of genes associated with plasticity in the frontal cortex, hippocampus, region encompassing the amygdala, as well as in blood, and displays similar effects relative to the SSRI fluoxetine in adult naïve rats. These genes encode immediate early genes (IEGs), translational regulators, and the neurodevelopmental marker Sema4g. Similar findings detected in brain regions and in blood provide a potential translational impact, and vortioxetine appears to consistently regulate signaling in these networks of neuroplasticity and developmental markers.

Use of Caenorhabditis elegans Gαq Chimeras to Detect G-Protein-Coupled Receptor Signals

G-protein-coupled receptors (GPCRs) activate heterotrimeric G-proteins (Gi-, Gs-, Gq-, or G12-like) to generate specific intracellular responses, depending on the receptor/G-protein coupling. The aim was to enable a majority of GPCRs to generate a predetermined output by signaling through a single G-protein-supported pathway. The authors focused on calcium responses as the output, then engineered Gαq to promote promiscuous receptor interactions. Starting with a human Gαq containing 5 Gαz residues in the C-terminal receptor recognition domain (hGαq/z5), they evaluated agonist-stimulated calcium responses for 33 diverse GPCRs (Gi-, Gs-, and Gq-coupled) and found 20 of 33 responders. In parallel, they tested Caenorhabditis elegans Gαq containing 5 or 9 C-terminal Gαz residues (cGαq/z5, cGαq/z9). Signal detection was enhanced with cGαq/z5 and cGαq/z9 (yielding 25/33 and 26/33 responders, respectively). In a separate study of Gαs-coupled receptors, the authors compared hGαq/s5 versus hGαq/s9, cGαq/s9, andcGαq/s21 and observed optimal function with cGαq/s9. Cotransfection of an engineered Gαq “cocktail” (cGαq/z5 plus cGαq/s9) provided a powerful and efficient screening platform. When the chimeras included N-terminal myristoylation sites (to promote membrane localization), calcium responses were sustained or improved, depending on the receptor. This approach toward a “universal functional assay” is particularly useful for orphan GPCRs whose signaling pathways are unknown.

In vivo and in vitro effects of vortioxetine on molecules associated with neuroplasticity

Neuroplasticity is fundamental for brain functions, abnormal changes of which are associated with mood disorders and cognitive impairment. Neuroplasticity can be affected by neuroactive medications and by aging. Vortioxetine, a multimodal antidepressant, has shown positive effects on cognitive functions in both pre-clinical and clinical studies. In rodent studies, vortioxetine increases glutamate neurotransmission, promotes dendritic branching and spine maturation, and elevates hippocampal expression of the activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) at the transcript level. The present study aims to assess the effects of vortioxetine on several neuroplasticity-related molecules in different experimental systems. Chronic (1 month) vortioxetine increased Arc/Arg3.1 protein levels in the cortical synaptosomes of young and middle-aged mice. In young mice, this was accompanied by an increase in actin-depolymerizing factor (ADF)/cofilin serine 3 phosphorylation without altering the total ADF/cofilin protein level, and an increase in the GluA1 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor phosphorylation at serine 845 (S845) without altering serine 831 (S831) GluA1 phosphorylation nor the total GluA1 protein level. Similar effects were detected in cultured rat hippocampal neurons: Acute vortioxetine increased S845 GluA1 phosphorylation without changing S831 GluA1 phosphorylation or the total GluA1 protein level. These changes were accompanied by an increase in α subunit of Ca2+/calmodulin-dependent kinase (CaMKIIα) phosphorylation (at threonine 286) without changing the total CaMKIIα protein level in cultured neurons. In addition, chronic (1 month) vortioxetine, but not fluoxetine, restored the age-associated reduction in Arc/Arg3.1 and c-Fos transcripts in the frontal cortex of middle-aged mice. Taken together, these results demonstrated that vortioxetine modulates molecular targets that are related to neuroplasticity.

Gene expression profiles in relation to tension and dissociation in borderline personality disorder.

The biological underpinnings of borderline personality disorder (BPD) and its psychopathology including states of aversive tension and dissociation is poorly understood. Our goal was to examine transcriptional changes associated with states of tension or dissociation within individual patients in a pilot study. Dissociation is not only a critical symptom of BPD but has also been associated with higher risk for self-mutilation and depression. We conducted a whole blood gene expression profile analysis using quantitative PCR in 31 female inpatients with BPD. For each individual, two samples were drawn during a state of high tension and dissociation, while two samples were drawn at non-tension states. There was no association between gene expression and tension states. However, we could show that Interleukin-6 was positively correlated to dissociation scores, whereas Guanine nucleotide-binding protein G(s) subunit alpha isoforms, Mitogen-activated protein kinase 3 and 8, Guanine nucleotide-binding protein G(i) subunit alpha-2, Beta-arrestin-1 and 2, and Cyclic AMP-responsive element-binding protein were negatively correlated to dissociation. Our data point to a potential association of dissociation levels with the expression of genes involved in immune system regulation as well as cellular signalling/second-messenger systems. Major limitations of the study are the the possibly heterogeneous cell proportions in whole blood and the heterogeneous medication.

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.