GABAB receptor cell-surface export is controlled by an endoplasmic reticulum gatekeeper.

Endoplasmic reticulum (ER) release and cell-surface export of many G protein-coupled receptors (GPCRs) are tightly regulated. For gamma-aminobutyric acid (GABA)B receptors of GABA, the major mammalian inhibitory neurotransmitter, the ligand-binding GB1 subunit is maintained in the ER by unknown mechanisms in the absence of hetero-dimerization with the GB2 subunit. We report that GB1 retention is regulated by a specific gatekeeper, PRAF2. This ER resident transmembrane protein binds to GB1, preventing its progression in the biosynthetic pathway. GB1 release occurs upon competitive displacement from PRAF2 by GB2. PRAF2 concentration, relative to that of GB1 and GB2, tightly controls cell-surface receptor density and controls GABAB function in neurons. Experimental perturbation of PRAF2 levels in vivo caused marked hyperactivity disorders in mice. These data reveal an unanticipated major impact of specific ER gatekeepers on GPCR function and identify PRAF2 as a new molecular target with therapeutic potential for psychiatric and neurological diseases involving GABAB function.

Genetic and functional analyses demonstrate a role for abnormal glycinergic signaling in autism.

Autism spectrum disorder (ASD) is a common neurodevelopmental condition characterized by marked genetic heterogeneity. Recent studies of rare structural and sequence variants have identified hundreds of loci involved in ASD, but our knowledge of the overall genetic architecture and the underlying pathophysiological mechanisms remains incomplete. Glycine receptors (GlyRs) are ligand-gated chloride channels that mediate inhibitory neurotransmission in the adult nervous system but exert an excitatory action in immature neurons. GlyRs containing the α2 subunit are highly expressed in the embryonic brain, where they promote cortical interneuron migration and the generation of excitatory projection neurons. We previously identified a rare microdeletion of the X-linked gene GLRA2, encoding the GlyR α2 subunit, in a boy with autism. The microdeletion removes the terminal exons of the gene (GLRA2(Δex8-9)). Here, we sequenced 400 males with ASD and identified one de novo missense mutation, p.R153Q, absent from controls. In vitro functional analysis demonstrated that the GLRA2(Δex8)(-)(9) protein failed to localize to the cell membrane, while the R153Q mutation impaired surface expression and markedly reduced sensitivity to glycine. Very recently, an additional de novo missense mutation (p.N136S) was reported in a boy with ASD, and we show that this mutation also reduced cell-surface expression and glycine sensitivity. Targeted glra2 knockdown in zebrafish induced severe axon-branching defects, rescued by injection of wild type but not GLRA2(Δex8-9) or R153Q transcripts, providing further evidence for their loss-of-function effect. Glra2 knockout mice exhibited deficits in object recognition memory and impaired long-term potentiation in the prefrontal cortex. Taken together, these results implicate GLRA2 in non-syndromic ASD, unveil a novel role for GLRA2 in synaptic plasticity and learning and memory, and link altered glycinergic signaling to social and cognitive impairments.

Brain organic cation transporter 2 controls response and vulnerability to stress and GSK3ß signaling.

Interactions between genetic and environmental factors, like exposure to stress, have an important role in the pathogenesis of mood-related psychiatric disorders, such as major depressive disorder. The polyspecific organic cation transporters (OCTs) were shown previously to be sensitive to the stress hormone corticosterone in vitro, suggesting that these transporters might have a physiologic role in the response to stress. Here, we report that OCT2 is expressed in several stress-related circuits in the brain and along the hypothalamic-pituitary-adrenocortical (HPA) axis. Genetic deletion of OCT2 in mice enhanced hormonal response to acute stress and impaired HPA function without altering adrenal sensitivity to adrenocorticotropic hormone (ACTH). As a consequence, OCT2(-/-) mice were potently more sensitive to the action of unpredictable chronic mild stress (UCMS) on depression-related behaviors involving self-care, spatial memory, social interaction and stress-sensitive spontaneous behavior. The functional state of the glycogen synthase kinase-3ß (GSK3ß) signaling pathway, highly responsive to acute stress, was altered in the hippocampus of OCT2(-/-) mice. In vivo pharmacology and western blot experiments argue for increased serotonin tonus as a main mechanism for impaired GSK3ß signaling in OCT2(-/-) mice brain during acute response to stress. Our findings identify OCT2 as an important determinant of the response to stress in the brain, suggesting that in humans OCT2 mutations or blockade by certain therapeutic drugs could interfere with HPA axis function and enhance vulnerability to repeated adverse events leading to stress-related disorders.

The absence of VGLUT3 predisposes to cocaine abuse by increasing dopamine and glutamate signaling in the nucleus accumbens.

Tonically active cholinergic interneurons (TANs) from the nucleus accumbens (NAc) are centrally involved in reward behavior. TANs express a vesicular glutamate transporter referred to as VGLUT3 and thus use both acetylcholine and glutamate as neurotransmitters. The respective roles of each transmitter in the regulation of reward and addiction are still unknown. In this study, we showed that disruption of the gene that encodes VGLUT3 (Slc17a8) markedly increased cocaine self-administration in mice. Concomitantly, the amount of dopamine (DA) release was strongly augmented in the NAc of VGLUT3(-/-) mice because of a lack of signaling by metabotropic glutamate receptors. Furthermore, dendritic spines and glutamatergic synaptic transmission on medium spiny neurons were increased in the NAc of VGLUT3(-/-) mice. Increased DA and glutamate signaling in the NAc are hallmarks of addiction. Our study shows that TANs use glutamate to reduce DA release and decrease reinforcing properties of cocaine in mice. Interestingly, we also observed an increased frequency of rare variations in SLC17A8 in a cohort of severe drug abusers compared with controls. Our findings identify VGLUT3 as an unexpected regulator of drug abuse.

In vivo occupancy of dopamine D3 receptors by antagonists produces neurochemical and behavioral effects of potential relevance to attention-deficit-hyperactivity disorder.

Dopamine D(3) receptors have eluded definitive linkage to neurologic and psychiatric disorders since their cloning over 20 years ago. We report a new method that does not employ a radiolabel for simultaneously defining in vivo receptor occupancy of D(3) and D(2) receptors in rat brain after systemic dosing using the tracer epidepride (N-[[(2S)-1-ethylpyrrolidin-2-yl]methyl]-5-iodo-2,3-dimethoxybenzamide). Decreases in epidepride binding in lobule 9 of cerebellum (rich in D(3) receptors) were compared with nonspecific binding in the lateral cerebellum. The in vivo occupancy of the dopamine D(3) receptors was dose dependently increased by SB-277011A (trans-N-[4-[2-(6-cyano-1,2,3,4-tetrahydroisoquinolin-2-yl)ethyl]cyclohexyl]-4-quinolinecarboxamide) and U99194 (2,3-dihydro-5,6-dimethoxy- N,N-dipropyl-1H-inden-2-amine). Both antagonists increased extracellular levels of acetylcholine (ACh) in the medial prefrontal cortex of rats and modified brain-tissue levels of ACh and choline. Consistent with these findings, the D(3) receptor antagonists enhanced the acquisition of learning of rats either alone or in the presence of the norepinephrine uptake blocker reboxetine as with the attention-deficit-hyperactivity disorder (ADHD) drug methylphenidate. Like reboxetine, the D(3) receptor antagonists also prevented deficits induced by scopolamine in object recognition memory of rats. Mice in which the dopamine transporter (DAT) has been deleted exhibit hyperactivity that is normalized by compounds that are effective in the treatment of ADHD. Both D(3) receptor antagonists decreased the hyperactivity of DAT(-/-) mice without affecting the activity of wild type controls. The present findings indicate that dopamine D(3) receptor antagonists engender cognition-enhancing and hyperactivity-dampening effects. Thus, D(3) receptor blockade could be considered as a novel treatment approach for cognitive deficits and hyperactivity syndromes, including those observed in ADHD.

Organic cation transporter 2 controls brain norepinephrine and serotonin clearance and antidepressant response.

High-affinity transporters for norepinephrine (NE) and serotonin (5-HT), which ensure neurotransmitter clearance at the synapse, are the principal targets of widely used antidepressant drugs. Antidepressants targeting these high-affinity transporters, however, do not provide positive treatment outcomes for all patients. Other monoamine transport systems, with lower affinity, have been detected in the brain, but their role is largely unknown. Here we report that OCT2, a member of the polyspecific organic cation transporter (OCT) family, is expressed notably in the limbic system and implicated in anxiety and depression-related behaviors in the mouse. Genetic deletion of OCT2 in mice produced a significant reduction in brain tissue concentrations of NE and 5-HT and in ex vivo uptake of both these neurotransmitters in the presence of the dual 5-HT-NE transport blocker, venlafaxine. In vivo clearance of NE and 5-HT evaluated using microiontophoretic electrophysiology was diminished in the hippocampus of OCT2(-/-) mice in the presence of venlafaxine, thereby affecting postsynaptic neuronal activity. OCT2(-/-) mice displayed an altered sensitivity to acute treatments with NE- and/or 5-HT-selective transport blockers in the forced-swim test. Moreover, the mutant mice were insensitive to long-term venlafaxine treatment in a more realistic, corticosterone-induced, chronic depression model. Our findings identify OCT2 as an important postsynaptic determinant of aminergic tonus and mood-related behaviors and a potential pharmacological target for mood disorders therapy.

Glycine receptor beta-subunit gene mutation in spastic mouse associated with LINE-1 element insertion.

Congenital myoclonus is a widespread neurologic disorder characterized by hyperexcitability, muscular spasticity and myoclonus associated with marked reduction in neural glycine binding sites. The recessive mouse mutation spastic (spa) is a prototype of inherited myoclonus. Here we show that defects in the gene encoding the beta-subunit of the glycine receptor (Glrb) underlie spa: Glrb maps to the same region of mouse chromosome 3 as spa, and Glrb mRNA is markedly reduced throughout brains of spa mice, most likely as a result of an insertional mutation of a 7.1 kilobase LINE-1 element within intron 6 of Glrb. These results provide evidence that Glrb is necessary for postsynaptic expression of glycine receptor complexes, and suggest Glrb as a candidate gene for inherited myoclonus in other species.

Anterior pituitary hypoplasia and dwarfism in mice lacking the dopamine transporter.

Deletion of the dopamine transporter (DAT) results in increased dopaminergic tone, anterior pituitary hypoplasia, dwarfism, and an inability to lactate. DAT elimination alters the spatial distribution and dramatically reduces the numbers of lactotrophs and somatotrophs in the pituitary. Despite having normal circulating levels of growth hormone and prolactin in blood, hypoplastic glands from DAT-/- mice fail to respond to secretagog stimulation. The effects of DAT deletion on pituitary function result from elevated DA levels that down-regulate the lactotroph D2 DA receptors and depress hypothalamic growth hormone-releasing hormone content. These results reveal an unexpected and important role or DA in the control of developmental events in the pituitary gland and assign a critical role for hypothalamic DA reuptake in regulating these events.

Cocaine self-administration in dopamine-transporter knockout mice.

The plasma membrane dopamine transporter (DAT) is responsible for clearing dopamine from the synapse. Cocaine blockade of DAT leads to increased extracellular dopamine, an effect widely considered to be the primary cause of the reinforcing and addictive properties of cocaine. In this study we tested whether these properties are limited to the dopaminergic system in mice lacking DAT. In the absence of DAT, these mice exhibit high levels of extracellular dopamine, but paradoxically still self-administer cocaine. Mapping of the sites of cocaine binding and neuronal activation suggests an involvement of serotonergic brain regions in this response. These results demonstrate that the interaction of cocaine with targets other than DAT, possibly the serotonin transporter, can initiate and sustain cocaine self-administration in these mice.

Neurochemical characterization of pathways expressing plasma membrane monoamine transporter in the rat brain.

Neurotransmitter transporters play an important role in the control of synaptic transmission by ensuring the clearance of transmitters liberated in the synaptic cleft. In the case of monoaminergic neurotransmitters, this clearance is carried out by high-affinity reuptake transporters located in the plasma membrane of the presynaptic terminals. Recently plasma membrane monoamine transporter (PMAT), a transporter from the SLC29 (equilibrative nucleoside transporter) family, was shown to transport in vitro monoaminergic neurotransmitters, in particular dopamine and serotonin, nearly as efficiently as the high-affinity transporters. This transporter, well expressed in CNS, represents an interesting candidate for the control and modulation of aminergic pathways. We performed an extensive study of the distribution of PMAT in the rat brain. Our results highlight PMAT expression in brain regions which play a pivotal role in significant CNS functions and human neuropathologies. Using in situ hybridization immunohistochemistry co-labeling, PMAT mRNA was found in various neuron subtypes, including glutamatergic neurons of the hippocampus, mitral cells of the olfactory bulbs and GABAergic neurons in the substantia nigra pars reticulata and hypothalamus. Paradoxically, rat PMAT mRNA was found in some but not all monoaminergic nuclei. It was on the contrary predominantly expressed in major cholinergic groups throughout the brain, including brainstem motor nuclei, components of the basal forebrain cholinergic system and cholinergic interneurons of the striatum. These systems, implicated in locomotion, associative and spatial memory and reward-related learning, are disrupted at early stages of Parkinson's and Alzheimer's disease. Taken together, our observations support a role for PMAT in monoamine uptake in cholinergic neurons.

Calcineurin (protein phosphatase 2B) is involved in the mechanisms of action of antidepressants.

Calcineurin (PP2B) is a Ca(2+)-dependent protein phosphatase enriched in the brain that takes part in intracellular signaling pathways regulating synaptic plasticity and neuronal functions. Calcineurin-dependent pathways are important for complex brain functions such as learning and memory. More recently, they have been suggested to play a role in the processing of emotional information. The aim of this study was to investigate whether calcineurin may be involved in the effect of antidepressants. We first found that chronic antidepressant treatment in mice leads to an increase of calcineurin levels in the hippocampus. We then studied the behavioral and molecular responses to fluoxetine of mice with a genetic overactivation of calcineurin in the hippocampus (constitutively-activated calcineurin transgenic mouse line #98, CN98 mice). We observed that CN98 mice are more sensitive to the behavioral effect of fluoxetine and desipramine tested in the tail suspension test. Moreover, the basal expression of growth factor brain-derived neurotrophic factor and subunit 1 of AMPA glutamate receptor, GluR1, both of which are modified after chronic antidepressant administration, are altered in the hippocampus of CN98 mice. These results suggest that calcineurin-dependent dephosphorylation plays an important role in the mechanisms of action of antidepressants, providing a new starting point for developing improved therapeutic treatments for depression.

The NH(2)-terminus of norepinephrine transporter contains a basolateral localization signal for epithelial cells.

When expressed in epithelial cells, dopamine transporter (DAT) was detected predominantly in the apical plasma membrane, whereas norepinephrine transporter (NET) was found in the basolateral membrane, despite 67% overall amino acid sequence identity. To identify possible localization signals responsible for this difference, DAT-NET chimeras were expressed in MDCK cells and localized by immunocytochemistry and transport assays. The results suggested that localization of these transporters in MDCK cells depends on their highly divergent NH(2)-terminal regions. Deletion of the first 58 amino acids of DAT (preceding TM1) did not change its apical localization. However, the replacement of that region with corresponding sequence from NET resulted in localization of the chimeric protein to the basolateral membrane, suggesting that the NH(2)-terminus of NET, which contains two dileucine motifs, contains a basolateral localization signal. Mutation of these leucines to alanines in the context of a basolaterally localized NET/DAT chimera restored transporter localization to the apical membrane, indicating that the dileucine motifs are critical to the basolateral localization signal embodied within the NET NH(2)-terminal region. However, the same mutation in the context of wild-type NET did not disrupt basolateral localization, indicating the presence of additional signals in NET directing its basolateral localization within the plasma membrane.

Disrupted hippocampal neuregulin-1/ErbB3 signaling and dentate gyrus granule cell alterations in suicide.

Neuregulin-1 (NRG1) and ErbB receptors have been associated with psychopathology, and NRG1-ErbB3 signaling has been shown to increase hippocampal neurogenesis and induce antidepressant-like effects. In this study, we aimed to determine whether deficits in NRG1 or ErbBs might be present in the hippocampus of suicide completers. In well-characterized postmortem hippocampal samples from suicides and matched sudden-death controls, we assessed gene expression and methylation using qRT-PCR and EpiTYPER, respectively. Moreover, in hippocampal tissues stained with cresyl violet, stereology was used to quantify numbers of granule cells and of glia. Granule cell body size was examined with a nucleator probe, and granule cell layer volume with a Cavalieri probe. Unmedicated suicides showed sharply decreased hippocampal ErbB3 expression and decreased numbers of ErbB3-expressing granule cell neurons in the anterior dentate gyrus; a phenomenon seemingly reversed by antidepressant treatment. Furthermore, we found ErbB3 expression to be significantly decreased in the dentate gyrus of adult mice exposed to chronic social defeat stress. Taken together, these results reveal novel suicidal endophenotypes in the hippocampus, as well as a putative etiological mechanism underlying suicidality, and suggest that antidepressant or NRG1 treatment may reverse a potential deficit in anterior dentate gyrus granule cell neurons in individuals at risk of dying by suicide.

Disrupted hippocampal neuregulin-1/ErbB3 signaling and dentate gyrus granule cell alterations in suicide.

This corrects the article DOI: 10.1038/tp.2017.132.

Cocaine and amphetamine increase extracellular dopamine in the nucleus accumbens of mice lacking the dopamine transporter gene.

Behavioral and biochemical studies suggest that dopamine (DA) plays a role in the reinforcing and addictive properties of drugs of abuse. Recently, this hypothesis has been challenged on the basis of the observation that, in mice genetically lacking the plasma membrane dopamine transporter [DAT-knock out (DAT-KO)], cocaine maintained its reinforcing properties of being self-administered and inducing place preference, despite the failure to increase extracellular dopamine in the dorsal striatum. Here we report that, in DAT-KO mice, cocaine and amphetamine increase dialysate dopamine in the medial part of the nucleus accumbens. Moreover, reboxetine, a specific blocker of the noradrenaline transporter, increased DA in the nucleus accumbens of DAT-KO but not of wild-type mice; in contrast, GBR 12909, a specific blocker of the dopamine transporter, increased dialysate dopamine in the nucleus accumbens of wild-type but not of DAT-KO mice. These observations provide an explanation for the persistence of cocaine reinforcement in DAT-KO mice and support the hypothesis of a primary role of nucleus accumbens dopamine in drug reinforcement.

The existence of a second vesicular glutamate transporter specifies subpopulations of glutamatergic neurons.

Before their exocytotic release during stimulation of nerve terminals, nonpeptide neurotransmitters are loaded into synaptic vesicles by specific transporters. Recently, a protein initially identified as brain-specific Na(+)-dependent inorganic phosphate transporter I (BNPI) has been shown to represent a vesicular glutamate transporter (VGLUT1). In this study, we investigated whether a highly homologous "differentiation-associated Na(+)-dependent inorganic phosphate transporter" (DNPI) is involved in glutamatergic transmission. Vesicles isolated from BON cells expressing recombinant DNPI accumulated l-glutamate with bioenergetical and pharmacological characteristics identical to those displayed by VGLUT1 and by brain synaptic vesicles. Moreover, DNPI localized to synaptic vesicles, at synapses exhibiting classical excitatory features. DNPI thus represents a novel vesicular glutamate transporter (VGLUT2). The distributions of each VGLUT transcript in brain were highly complementary, with only a partial regional and cellular overlap. At the protein level, we could only detect either VGLUT1- or VGLUT2-expressing presynaptic boutons. The existence of two VGLUTs thus defines distinct subsets of glutamatergic neurons.

Molecular characterization of the dopamine transporter.

Neurotransmission, which represents chemical signalling between neurons, usually takes place at highly differentiated anatomical structures called synapses. To fulfill both the time and space confinements required for optimal neurotransmission, highly specialized proteins, known as transporters or uptake sites, occur and operate at the presynaptic plasma membrane. Using the energy provided by the Na+ gradient generated by the Na+/K(+)-transporting ATPase, these transporters reuptake the neurotransmitters soon after their release, thereby regulating their effective concentrations at the synaptic cleft and the availability of neurotransmitters for a time-dependent activation of both pre- and postsynaptic receptors. The key role these proteins play in normal neurotransmission is further emphasized when the physiological and social consequences of drugs that interfere with the function of these transporters, such as the psychostimulants (e.g. amphetamine and cocaine) or the widely prescribed antidepressant drugs, are considered. In this review, Bruno Giros and Marc Caron elaborate on the potential consequences of the recent molecular cloning of the dopamine and related transporters and summarize some of the interesting properties that are emerging from this growing family of Na(+)- and Cl(-)-dependent transporters.

Failure to find evidence for linkage or association between the dopamine D3 receptor gene and schizophrenia.

OBJECTIVE: This study was performed to assess the possible involvement of the dopamine D3 receptor gene (DRD3) in the etiology of schizophrenia. The authors' approach included a population study and a family study using both parametric (lod score) and nonparametric (affected pedigree member) methods of linkage analysis. METHOD: Two different DNA markers were studied at the DRD3 locus. The family study included 35 multiplex families of schizophrenic subjects for the linkage analyses. The population study involved 50 unrelated schizophrenic subjects and 50 normal comparison subjects from the same ethnic and geographic origin. RESULTS: Whichever clinical classification was used to define the pathological phenotype (schizophrenia or schizophrenia spectrum), the results of the lod score and affected pedigree member studies did not provide any evidence of linkage of the DRD3 gene to the illness. The negative results of the association study reinforce these results. CONCLUSIONS: The hypothesis that the DRD3 gene has a predisposing role in schizophrenia was not supported by these population and family studies. However, the possibility that this gene has a role in the etiology of the disease cannot be definitely excluded because of the intrinsic limitations of the methods of analysis and the number of subjects studied.

Cloning and functional characterization of a cocaine-sensitive dopamine transporter.

We report the cloning of a rat cDNA encoding a functional dopamine transporter. This cDNA, derived from an intron-containing gene, encodes a protein of 620 amino acids. Hydropathicity analysis of the protein sequence suggests the presence of 12 putative transmembrane domains. The protein displays considerable identity with transporters for noradrenaline and GABA (64 and 30%, respectively). Transient expression of the cDNA in COS7 cells directs the expression of dopamine uptake activity with appropriate pharmacology and in a sodium-dependent fashion. In situ hybridization reveals that the mRNA for this transporter is expressed in the substantia nigra and ventral tegmental area, regions that contain dopaminergic cell bodies.

Cloning of a functional vesicular GABA and glycine transporter by screening of genome databases.

The unc-47 locus of Caenorhabditis elegans has been suggested to encode a synaptic vesicle GABA transporter. Here we used hydropathy plot analysis to identify a candidate vesicular GABA transporter in genomic sequences derived from a region of the physical map comprising unc-47. A mouse homologue was identified and cloned from EST database information. In situ hybridization in rat brain revealed codistribution with both GABAergic and glycinergic neuronal markers. Moreover, expression in COS-7 and PC12 cells induced an intracellular, glycine-sensitive GABA uptake activity. These observations, consistent with previous data on GABA and glycine uptake by synaptic vesicles, demonstrate that the mouse clone encodes a vesicular inhibitory amino acid transporter.