Research Findings - Basic Neuroscience Research
PDZ binding of PICK1
The protein PICK1 (protein that interacts with C kinase 1) functions as a scaffolding or anchoring protein for the assembly of various protein partners, including protein kinase Calpha, AMPA receptor subunits 2-4, metabatropic glutamate receptors, transmembrane transporters, ion channels, and G protein-coupled receptors. Its binding to these varied partners can affect their level of surface plasma membrane expression in various cells, or, in some cases, their intracellular levels. PICK1 is highly expressed in the brain, including neuronal synapses. For binding, PICK1 possesses a single so-called "PDZ domain" near its N-terminus, consisting of amino acid residues 22-105, which provides a binding pocket for the PDZ-binding region of other proteins, primarily accommodating only several residues of the C-terminus of these proteins, with low micromolar affinity constants. A recent report by Dr. Gether and colleagues described the discovery of a small molecule known as FSC231, which serves as an inhibitor of the PICK1 PDZ domain. This molecule was found in a chemical library of approximately 44,000 compounds, using a competition assay for the inhibition of fluorescence of Oregon green-labeled DAT13 (a 13 amino acid C-terminus peptide of the DAT). This research has provided evidence for the disruption of the PICK1 PDZ-GluR2 complex by a small molecule with moderate binding affinity in the micromolar range. It may provide a starting point for the development of additional small molecule inhibitors, which may have the property of reducing hippocampal LTP induced by cocaine exposure and reversing the resulting redistribution of synaptic GluR subunits. Thorsen TS, Madsen KL, Rebola N, Rathje M, Anggono V, Bach A, Moreira I S, Stuhr-Hansen N, Dyhring T, Peters D, Beuming T, Huganir R, Weinstein H, Mulle C, Stromgaard K, Ronn LCB, Gether U. Identification of a small-molecule inhibitor of the PICK1 PDZ domain that inhibits hippocampal LTP and LTD. PNAS. 2010; 107(1): 413-418.
Characterization of Tunable Piperidine and Piperazine Carbamates as Inhibitors of Endocannabinoid Hydrolases
Monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) are two enzymes from the serine hydrolase superfamily that degrade the endocannabinoids 2-arachidonoylglycerol and anandamide, respectively. A group led by Dr. Benjamin Cravatt has recently discovered that MAGL and FAAH are both inhibited by carbamates bearing an N-piperidine/piperazine group. Piperidine/piperazine carbamates show excellent in vivo activity, raising brain endocannabinoid levels and producing CB1-dependent behavioral effects in mice, suggesting that they represent a promising class of inhibitors for studying the endogenous functions of MAGL and FAAH. Herein, they disclose a full account of the syntheses, structure-activity relationships, and inhibitory activities of piperidine/piperazine carbamates against members of the serine hydrolase family. These scaffolds can be tuned for MAGL-selective or dual MAGL-FAAH inhibition by the attachment of an appropriately substituted bisarylcarbinol or aryloxybenzyl moiety, respectively, on the piperidine/piperazine ring. Modifications to the piperidine/piperazine ring ablated inhibitory activity, suggesting a strict requirement for a six-membered ring to maintain potency. Long JZ, Jin X, Adibekian A, Li W, Cravatt BF. Characterization of tunable piperidine and piperazine carbamates as inhibitors of endocannabinoid hydrolases. J Med Chem. 2010; 53: 1830-1842.
Synthesis and Pharmacological Evaluation of Highly Potent [Dmt1]DALDA Analogs
Chemical structure variation of the highly potent and µ-opioid selective, peripheral analgesic, DALDA ([D-Arg2, Lys4] dermorphin-(1,4)-amide) led to the synthesis of a new compound [Dmt1] DALDA which had 12 fold higher affinity than DALDA at µ-opioid receptors, and a potency 180 fold higher. Authors had access to protected analogs of [Dmt1] DALDA which they used to synthesize gram quantities of three antioxidant peptides: SS-02 (Dmt-D-Arg-Phe-Lys-NH2). SS-31 (D-Arg-Dmt-Lys-Phe-NH2), and SS-20 (Phe-D-Arg-Phe-Lys-NH2), and then examined their bioactivity. Synthesis of these three peptides involved routinely used side chain protecting groups for amino acid building blocks. Opioid activities and potency varied at the at µ-opioid and δ-opioid receptors among these chemical structures. Reddy PA, Lewin AH, Schiller PW. Synthesis and pharmacological evaluation of highly potent [Dmt1] DALDA analogs. Adv Exp Med Biol. 2009; 611:473-474.
PET Imaging of Cortical Activation During Cocaine Self-Administration and Extinction
Dr. Leonard Howell and his colleagues at Emory University and Yerkes National Primate Research Center were among the first to use functional brain imaging to show cocaine-induced changes in brain activity during active drug taking in primates. Using PET imaging with O15-labeled water to quantify changes in cerebral blood flow, the PI and his colleagues studied the effects of cocaine administered noncontingently in nave monkeys, and the effects when the animals self-administered the drug or later, when saline was substituted for the cocaine. They found that noncontingent drug administration was associated with robust activation of the dorsolateral regions of the prefrontal cortex; by contrast, the effects of the drug when self-administered were to activate anterior cingulate cortex. During times when saline was substituted for cocaine, the stimuli previously paired with cocaine also induced a similar robust activation in prefrontal cortex. Dr. Howell and his colleagues interpret these findings to indicate that the effects of cocaine and associated cues extend further than the limbic system and utilize brain areas involved in cognitive processes. The recognition that neural circuits underlie the direct pharmacological and conditioned stimulus effects of cocaine may provide important new clues for the development of medications for the treatment of cocaine abuse. Howell LL, Votow JR, Goodman MM, Lindsey KP. Cortical activation during cocaine use and extinction in rhesus monkeys. Psychopharmacology. 2010; 208: 191-199.
Cannabinoid Receptor Agonist, THC, Inhibits Macrophage Migration to the Tat Protein of HIV-1
Macrophages and macrophage-like cells are important targets of HIV-1 infection at peripheral sites and in the central nervous system. After infection, these cells secrete a plethora of toxic factors, including the viral regulatory trans-activating protein (Tat). This protein is highly immunogenic and also serves as a potent chemoattractant for monocytes. In the present study, the exogenous cannabinoids THC and CP55940 were shown to significantly inhibit migration of human U937 macrophage-like cells to the Tat protein in a concentration-related manner. The CB1 receptor-selective agonist ACEA had no effect on Tat-mediated migration. In contrast, the CB2 receptor-selective agonist O-2137 exerted a concentration-related inhibition of U937 cell migration in response to Tat. Pharmacological blockage of CB1 receptor signaling using the antagonist (SR141716A had no effect on CP55940-mediated inhibition of macrophage migration to Tat, whereas treatment with the CB2 receptor antagonist SR144528 reversed the CP55940-mediated inhibition of migration. In addition, THC had no inhibitory effect on U937 migration to Tat after small interfering RNA knockdown of the CB2 receptor. Collectively, the pharmacological and biochemical knockdown data indicate that cannabinoid-mediated modulation of macrophage migration to the HIV-1 Tat protein is linked to the CB2 cannabinoid receptor. Furthermore, these results suggest that the CB2 cannabinoid receptor has potential to serve as a therapeutic target for ablation of HIV-1-associated untoward inflammatory response. Raborn ES, Cabral GA. Cannabinoid inhibition of macrophage migration to the trans-activating (Tat) protein of HIV-1 Is linked to the CB2 cannabinoid receptor. J Pharm Exp Ther. 2010; Apr; 333(1): 319-327.
Supraspinal Inactivation of Mitochondrial Superoxide Dismutase is a Source of Peroxynitrite in the Development of Morphine Antinociceptive Tolerance
Effective treatment of chronic pain with morphine is limited by decreases in the drug's analgesic action with chronic administration (antinociceptive tolerance). Because opioids are mainstays of pain management, restoring their efficacy has great clinical importance. It has been reported that formation of peroxynitrite in the dorsal horn of the spinal cord plays a critical role in the development of morphine antinociceptive tolerance and that nitration and enzymatic inactivation of mitochondrial superoxide dismutase (MnSOD) at that site provides a source for this nitroxidative species. These researchers report for the first time that antinociceptive tolerance in mice is also associated with the inactivation of MnSOD at supraspinal sites. Inactivation of MnSOD led to nitroxidative stress as evidenced by increased levels of products of oxidative DNA damage and activation of the nuclear factor poly (ADP-ribose) polymerase in whole brain homogenates. Co-administration of morphine with potent Mn porphyrin-based peroxynitrite scavengers, restored the enzymatic activity of MnSOD, (2) attenuated PN-derived nitroxidative stress, and (3) blocked the development of morphine-induced antinociceptive tolerance. A more lipophilic analogue was able to cross the blood-brain barrier and was about 30-fold more efficacious. Collectively, these data suggest that PN-mediated enzymatic inactivation of supraspinal MnSOD provides a source of nitroxidative stress, which in turn contributes to central sensitization associated with the development of morphine antinociceptive tolerance. Thus, PN-targeted therapeutics may have potential as adjuncts to opiates in pain management. Doyle T, Bryant L, Batinic-Haberle I, Little J, Cuzzocrea S, Masini E, Spasojevic I, Salvemini D. Supraspinal inactivation of mitochondrial superoxide dismutase is a source of peroxynitrite in the development of morphine antinociceptive tolerance. Neuroscience 2009; Dec. 164:702-710.
Opioids Activate Brain Analgesic Circuits through Cytochrome P450/Epoxygenase Signaling
Activation of μ opioid receptors by morphine in the brainstem and spinal cord produces analgesia, but the relevant post-receptor mechanisms are not known. In the brainstem, opioid-induced stimulation of descending circuits from the ventrolateral periaqueductal gray (PAG) and rostral ventromedial medulla (RVM) produces powerful inhibition of spinal nociceptive transmission. In the PAG, μ-opioid receptors activate these circuits by increasing a presynaptic, voltage-dependent potassium conductance that inhibits GABA release. Biochemical and electrophysiological studies have suggested that phospholipase A2 and its product, arachidonic acid, are important for such analgesic signaling. These researchers evaluated the arachidonate epoxygenase pathway, which uses P450s to produce up to four distinct epoxyeicosatrienoic acid isomers, as a brain μ opioid analgesic transduction mechanism. To assess the importance of brain cytochrome P450 (P450) activity in mu opioid analgesic action, these investigators generated mutant mice with brain neuron-specific reductions in P450 activity. These mice showed highly attenuated morphine antinociception compared with controls. Pharmacological inhibition of brain P450 arachidonate epoxygenases also blocked morphine antinociception in mice and rats. These findings indicate that a neuronal P450 epoxygenase mediates the pain-relieving properties of morphine. Conroy JL, Fang C, Gu J, Zeitlin SO, Yang W, Yang J, VanAlstine MA, Nalwalk JW, Albrecht PJ, Mazurkiewicz JE, Snyder-Keller A, Shan Z, Zhang SZ, Wentland MP, Behr M, Knapp BI, Bidlack JM, Zuiderveld OP, Leurs R, Ding X, Hough LB. Opioids activate brain analgesic circuits through cytochrome P450/epoxygenase signaling. Nat Neurosci. 2010; 13: 284-286.
The Beta-lactam Antibiotic, Ceftriaxone, Prevents Relapse to Cocaine Seeking by Restoring Glutamate Transport
The cystine-glutamate exchanger (xCT) is downregulated in the brain after chronic cocaine, resulting in reduced extracellular levels of nucleus accumbens glutamate. The importance of cocaine-induced loss of glutamate homeostasis is revealed by N-acetylcysteine restoring cystine-glutamate exchange and attenuating reinstatement to cocaine seeking. Another regulator of extracellular glutamate is the glial glutamate transporter (GLT-1). Dr. Kalivas and his colleagues hypothesized that cocaine self-administration reduces GLT-1 and that GLT-1 upregulation inhibits cocaine seeking. The authors measured [(3)H] glutamate uptake and protein expression of GLT-1 and xCT, the catalytic subunit of the cystine-glutamate exchanger, following cocaine self-administration and 3 weeks of extinction training. They also examined the affect of ceftriaxone (previously shown to increase GLT-1) and N-acetylcysteine treatment on the expression of GLT-1 and xCT. Ceftriaxone was also tested for the capacity to inhibit cue- and cocaine-induced relapse. Results indicated that self-administration reduced glutamate uptake and the expression of both GLT-1 and xCT. Ceftriaxone restored GLT-1 and xCT levels and prevented cue- and cocaine-induced reinstatement of drug seeking. N-acetylcysteine also restored GLT-1 and xCT levels. These results indicate that glutamate transport and cystine-glutamate exchange may be coregulated and provide further evidence that targeting glutamate homeostasis is a potential method for treating cocaine relapse. Knackstedt LA, Melendez RI, Kalivas PW. Ceftriaxone restores glutamate homeostasis and prevents relapse to cocaine seeking. Biol Psychiatry. 2010 Jan 1; 67(1): 81-84.
Benzodiazepines Promote Addiction by Increasing VTA Dopamine Firing through Disinhibition of Interneuron GABA Release
Addictive drugs increase the levels of dopamine and also trigger long-lasting synaptic adaptations in the mesolimbic reward system that ultimately may induce the pathological behaviour. Addictive drugs can be classified into three groups according to the mechanism through which they increase mesolimbic dopamine; 1) indirectly increasing dopamine neuron firing rates by reducing inhibitory neuron input, 2) directly activating dopamine neurons, or 3) altering dopamine transporter function. However, it is unclear whether these mechanisms can account for the addiction liability of benzodiazepines. This paper shows that benzodiazepines indirectly increase firing of dopamine neurons of the ventral tegmental area through the positive modulation of GABA(A) (gamma-aminobutyric acid type A) receptors in nearby interneurons. Such disinhibition, which relies on alpha1-containing GABA(A) receptors expressed in these cells, triggers drug-evoked synaptic plasticity in excitatory afferents onto dopamine neurons and underlies drug reinforcement. Taken together, the data provide evidence that benzodiazepines share defining pharmacological features of addictive drugs through cell-type-specific expression of alpha1-containing GABA(A) receptors in the ventral tegmental area. The data also indicate that subunit-selective benzodiazepines sparing alpha1 may be devoid of addiction liability. Tan KR, Brown M, Laboube G, Yvon C, Creton C, Fritschy JM, Rudolph U, Lscher C. Neural bases for addictive properties of benzodiazepines. Nature 2010 Feb 11; 463(7282): 769-774.
Muscarinic Acetylcholine Type M2 and GABA(B) R2 Receptors Form Novel Heterodimer Receptors that Enhance Cholinergic Signaling in the Brain
Chronic stimulation of muscarinic M2 receptors [M(2)R] has previously been shown to promote internalization of G-protein-activated inwardly rectifying potassium channels (GIRKs) which results in loss of function. However, emerging evidence is showing that G-protein-coupled receptors (GPCRs) can form heterodimeric receptor complexes that generate new GPCR signaling properties within the brain. This paper now reports that coexpression of GABA(B) R2 receptors (GBR2s) rescues both surface expression and function of muscarinic M2 receptors, including M(2)R-induced activation of G-protein-activated inwardly rectifying potassium channels (GIRKs). GBR2 showed significant association with M(2)R at the plasma membrane but not with other GPCRs (such as M(1)R, mu-opioid receptor), as detected by fluorescence resonance energy transfer (FRET). Unique regions of the proximal C-terminal domains of GBR2 and M(2)R mediate specific binding between M(2)R and GBR2. In the brain, GBR2, but not GBR1, biochemically coprecipitates with M(2)R and overlaps with M(2)R expression in cortical neurons. This novel heteromeric association between M(2)R and GBR2 provides a possible mechanism for altering muscarinic signaling in the brain and represents a previously unrecognized role for GBR2. Boyer SB, Clancy SM, Terunuma M, Revilla-Sanchez R, Thomas SM, Moss SJ, Slesinger PA. Direct interaction of GABAB receptors with M2 muscarinic receptors enhances muscarinic signaling. J Neurosci 2009 Dec 16; 29(50): 15796-15809.
Glutamate or Norepinephrine-mediated Synaptic Plasticity within the Extended Amygdala Involve Different Cellular Mechanisms and are Differentially Affected in Response to Stress
Changes in the amygdala have been proposed as potential mechanisms underlying the development of addictive behaviors. Long-term depression (LTD), a form of synaptic plasticity, is an important synaptic mechanism for limiting excitatory influence over circuits subserving cognitive and emotional behavior. A major means of LTD induction is through the recruitment of signaling via G-protein coupled receptors [GPCR] activated by norepinephrine (NE) and glutamate. Receptors from these transmitter families have been proposed to converge on a common postsynaptic LTD mechanism to produce similar alterations in glutamate synapse efficacy. This paper reports that in the bed nucleus of the stria terminalis (BNST), a structure within the extended amygdala, recruitment of G-protein coupled receptors by glutamate or NE initiates mechanistically distinct forms of postsynaptically maintained LTD. Furthermore, the LTDs produced are differentially regulated by stress exposure. In particular, although both glutamate [mGluR5] and norepinephrine [alpha(1)-adrenergic receptor (AR)]-dependent LTDs involve postsynaptic endocytosis, norepinephrine-initiated LTD exclusively involves modulation of signaling through calcium-permeable AMPA receptors. Further, norepinephrine, but not glutamate, dependent LTD is disrupted by restraint stress. These data suggest that in the BNST, NE- and glutamate-activated GPCR pathways differentially tune glutamate synapse efficacy in response to stress. McElligott ZA, Klug JR, Nobis WP, Patel S, Grueter BA, Kash TL, Winder DG. Distinct forms of Gq-receptor-dependent plasticity of excitatory transmission in the BNST are differentially affected by stress. Proc Natl Acad Sci USA 2010 Feb 2; 107(5): 2271-2276.
Differential Modulation of Mu- and Delta-Opioid Receptor Agonists by Endogenous RGS4 Protein in SH-SY5Y Cells
Regulator of G-protein signaling (RGS) proteins are a family of molecules that control the duration of G protein signaling. A variety of RGS proteins are thought to modulate opioid receptor signaling. Here Dr. Traynor of University of Michigan Medical School and his research team show that RGS4 is abundantly expressed in human neuroblastoma SH-SY5Y cells that endogenously express mu- and delta-opioid receptors and test the hypothesis that the activity of opioids in these cells is modulated by RGS4. Endogenous RGS4 protein was reduced by approximately 90% in SH-SY5Y cells stably expressing short hairpin RNA specifically targeted to RGS4. In these cells, the potency and maximal effect of delta-opioid receptor agonist (SNC80)-mediated inhibition of forskolin-stimulated cAMP accumulation was greater than that seen in control cells. Transient transfection of a stable RGS4 mutant (HA-RGS4C2S) reversed this effect. Further, the delta opiate agonist increased MAPK activation in cells with less RGS4, but there was no change in the mu-opioid (morphine) response at adenylyl cyclase or MAPK. FLAG-tagged opioid receptors and HA-RGS4C2S were transiently expressed in HEK293T cells, and co-immunoprecipitation experiments showed that the delta-opioid receptor but not the mu-opioid receptor could be precipitated together with the stable RGS4. The use of delta- and mu-opioid receptor chimeras indicated that the C-tail and third intracellular domain of the delta-opioid receptor could be the sites of interaction with RGS4. The findings demonstrate a role for endogenous RGS4 protein in modulating delta-opioid receptor signaling in SH-SY5Y cells and provide evidence for a receptor-specific effect of RGS4. Wang, Q, Liu-Chen, L-Y, Traynor, JR. Differential modulation of mu- and delta-opioid receptor agonists by endogenous RGS4 protein in SH-SY5Y cells. J Biol Chem 2009; 284(27): 18357-18367.
Mu-Opioid Receptor Endocytosis Prevents Adaptations in Ventral Tegmental Area GABA Transmission Induced During Naloxone-Precipitated Morphine Withdrawal
Chronic morphine drives adaptations in synaptic transmission thought to underlie opiate dependence. Here Dr. Whistler and colleagues at the Ernest Gallo Clinic and Research Center examine the role of mu-opioid receptor (MOR) trafficking in one of these adaptations, specifically, changes in GABA transmission in the ventral tegmental area (VTA). To address this question, they used a knock-in mouse, RMOR (for recycling MOR), in which genetic change in the MOR promotes morphine-induced receptor desensitization and endocytosis in GABA interneurons of the VTA. In wild-type mice repeated injections of morphine (10 mg/kg, s.c., twice daily for 5 d), induced a cAMP-dependent increase in the probability of GABA release onto VTA dopamine neurons. The increased GABA release frequency correlated with physical dependence on morphine measured by counting somatic signs of morphine withdrawal, such as, tremors, jumps, rears, wet-dog shakes, and grooming behavior precipitated by subcutaneous administration of naloxone. This adaptation in GABA release was prevented in RMOR mice given the same morphine treatment, implicating MOR trafficking in this morphine-induced change in plasticity. Importantly, treatment with the cAMP activity inhibitor rp-cAMPS [(R)-adenosine, cyclic 3',5'-(hydrogenphosphorothioate) triethylammonium] directly to the VTA attenuated somatic withdrawal signs to systemic morphine produced by intra-VTA naloxone, directly linking enhanced cAMP-driven GABA release to naloxone-precipitated morphine withdrawal in the VTA. Madhavan A, He L, Stuber GD, Bonci A, Whistler JL. Mu-opioid receptor endocytosis prevents adaptations in ventral tegmental area GABA transmission induced during naloxone-precipitated morphine withdrawal. J Neurosci. 2010 Mar 3; 30(9): 3276-3286.
The Quantal Size of Dopamine Release is Regulated by Glutamate in Dopamine Neurons that Co-Release Glutamate
Recent evidence strongly suggests that a subset of dopamine neurons in the ventral tegmental area (VTA) release not only dopamine but also release the excitatory neurotransmitter glutamate. To assess a physiological role for glutamate co-release, these researchers disrupted the expression of vesicular glutamate transporter 2 selectively in dopamine neurons. This conditional knockout abolishes glutamate release from midbrain dopamine neurons in culture and severely reduces their excitatory synaptic output in mesoaccumbens slices. Baseline motor behavior is not affected, but stimulation of locomotor activity by cocaine is impaired, along with a selective reduction of dopamine stores in the projection of VTA neurons to ventral striatum. Glutamate co-entry promotes dopamine storage by increasing the pH gradient that drives vesicular monoamine transport. Low concentrations of glutamate acidify synaptic vesicles more slowly, but to a greater extent, than equimolar Cl-, indicating a distinct, presynaptic mechanism to regulate dopamine quantal size Hnasko TS, Chuhma N, Zhang H, Goh GY, Sulzer D, Palmiter RD, Rayport S, Edwards RH. Vesicular glutamate transport promotes dopamine storage and glutamate corelease in vivo. Neuron 2010 Mar 11; 65(5): 643-656.
Cocaine-induced Regulation of Striatal Dopamine Transporter uptake is Different in Rats with High and Low Locomotor Responsivity to Cocaine
While cocaine use is a serious public health issue, only 10-15% of initial users develop addiction to the drug. Previously, Dr. Zahniser had identified SD rats that responded with increased (HCR, high cocaine responders) or decreases (LCR, low cocaine responders) and sought to identify the neurochemical bases of their individual differences. She found that 30 min after a single injection of cocaine, the uptake of DA into striatal synaptosomes of HCRs was greater than that of LCRs. She extended the observation time in this study and found that HCRs exhibited a marked initial locomotor activation that returned to baseline by 120 min post-injection. While LCRs exhibited a >50% lower maximal locomotor response, this increase was sustained, lasting approximately 33% longer than in HCRs. At 25 min post-cocaine, maximal velocity (of [(3)H]DA uptake was significantly higher by 25% in HCRs than LCRs, with no difference in affinity. Despite the difference in the rate of uptake, however, DAT surface expression did not differ between LCRs and HCRs. These findings suggest that, compared to LCRs, HCRs have an enhanced ability to rapidly up-regulate DAT function in response to acute cocaine, which may be related to their cocaine-induced locomotor activation. Mandt BH, Zahniser NR. Low and high cocaine locomotor responding male Sprague-Dawley rats differ in rapid cocaine-induced regulation of striatal dopamine transporter function. Neuropharmacology. 2010 Mar; 58(3): 605-612. Epub 2009 Dec 4.
Meta-analysis of 15 Genome-wide Linkage Scans of Smoking Behavior
A genetic contribution to smoking behavior is well-established. To identify loci that increase the risk for smoking behavior, many genome-wide linkage scans have been performed with various smoking behavior assessments. Numerous putative susceptibility loci have been identified, but only a few of these were replicated in independent studies. Dr. Gelernter and colleagues used genome search meta-analysis (GSMA) to identify risk loci by pooling all available independent genome scan results on smoking behavior. Additionally, to minimize locus heterogeneity, subgroup analyses of the smoking behavior assessed by the Fagerstrom Test for Nicotine Dependence (FTND) and maximum number of cigarettes smoked in a 24-hour period (MaxCigs24) were carried out. Samples of European ancestry were also analyzed separately. A total number of 15 genome scan results were available for analysis, including 3404 families with 10,253 subjects. Overall, the primary GSMA across all smoking behavior identified a genome-wide suggestive linkage in chromosome 17q24.3-q25.3 (p(SR) = .001). A secondary analysis of FTND in European-ancestry samples (625 families with 1878 subjects) detected a genome-wide suggestive linkage in 5q33.1-5q35.2 (p(SR) = .0076). Subgroup analysis of MaxCigs24 (966 families with 3273 subjects) identified a genome-wide significant linkage in 20q13.12-q13.32 (p(SR) = .00041, p(OR) = .048), where a strongly supported nicotine dependence candidate gene, CHRNA4, is located. The regions identified in the current study deserve close attention and will be helpful for candidate gene identification or target re-sequencing studies in the future. Han S, Gelernter L, Luo X, Yang BZ. Biol Psychiatry. Meta-analysis of 15 genome-wide linkage scans of smoking behavior. 2010 Jan 1; 67(1):12-19.
Histone Dimethylation is Essential for Cocaine-induced Neuronal and Behavioral Plasticity
Cocaine can induce behavioral changes by altering expression of neuronal genes through mechanisms that have been somewhat unclear. One possible mechanism is to modify histone proteins associated with DNA to form chromatin, the structural scaffold for chromosome. By modifying and thereby altering the structural conformation of histones, protein factors that turn off or on a gene can gain access or be prevented from gaining acesss to the DNA sequence encoding a gene. Dr. Nestler, Dr. Greengard, and colleagues observed that histone methylation levels are reduced in the nucleus accumbens (NAc) of rodents. To further explore this phenomenon, Dr. Nestler investigated the gene expression levels of the histone dimethyltransferases and demethylases that regulate this chromatin modification and found that levels of the G9a and GLP histone dimethyltransferases are downregulated upon cocaine administration. Dr. Nestler then looked at the effect of G9a manipulation in the NAc on the behavioral effects of cocaine. He found that overexpression of G9a decreased the rewarding properties of cocaine, while knockdown of G9a increased the rewarding properties of cocaine. Dr. Nestler showed that these behavioral changes were correlated with concomitant changes in G9a levels and global histone dimethylation. Looking upstream of G9a, Dr. Nestler presented evidence that repeated cocaine exposure increases the levels of the transcription factor deltafosB leading to decreased G9a levels. Looking downstream, Dr. Nestler showed that many of the genomic targets of histone dimethylation are involved in regulation of dendritic plasticity and further that dendritic spine density is altered by G9a levels. Overall Dr. Nestler and colleagues have elucidated an elegant multistep molecular pathway leading from repeated cocaine exposure to deltafosB activation, downregulation of G9a, and reduction in global histone demethylation levels. Histone demethylation is normally associated with gene silencing, so decreased histone methylation leads to increased expression of genes that regulate dendritic plasticity. This change in gene expression leads to increased dendritic spine density and ultimately increased behavioral preference for cocaine. Small molecules that target the activities of histone demethylases or histone dimethyltransferases could have potential efficacy as therapeutic agents for treating cocaine addiction. Maze I, Covington HE 3rd, Dietz DM, LaPlant Q, Renthal W, Russo SJ, Mechanic M, Mouzon E, Neve RL, Haggarty SJ, Ren Y, Sampath SC, Hurd YL, Greengard P, Tarakhovsky A, Schaefer A, Nestler EJ. Essential role of the histone methyltransferase G9a in cocaine-induced plasticity. Science. 2010; 327(5962): 213-216.
Functional Impact of a Single-nucleotide Polymorphism in the OPRD1 Promoter Region
The delta-opioid receptor mediates rewarding effects of many substances of abuse. Dr. Zhang and colleagues reported an increased frequency of the minor G-allele of single-nucleotide polymorphism (SNP) rs569356 (the only variant identified so far in the promoter region of the delta-opioid receptor gene (OPRD1)) in subjects with opioid dependence. In this study, Dr. Zhang and colleagues examined the functional significance of this variant. OPRD1 promoter region harboring SNP rs569356 was amplified by PCR and inserted into a firefly luciferase reporter vector. HEK293 cells were co-transfected with these constructs and a renilla luciferase vector to control for transfection efficiency. Expression of firefly luciferase (driven by the OPRD1 promoter) was measured by a dual luciferase reporter assay and normalized by renilla luciferase expression. Moreover, alleles altering expression were further assessed for binding of human brain nuclear proteins by electrophoretic mobility shift assay (EMSA). The minor G-allele was associated with significantly greater expression levels of firefly luciferase than the major A-allele of SNP rs569356 (P=0.003). EMSA also showed specific gel shift bands, suggesting that SNP rs569356 is situated in the binding site of potential transcription factors. These results suggest that the minor G-allele of SNP rs569356 may enhance transcription factor binding and increase OPRD1 expression. Zhang H, Gelernter J, Gruen JR, Kranzler HR, Herman AI, Simen AA. Functional impact of a single-nucleotide polymorphism in the OPRD1 promoter region. J Hum Genet. 2010 Mar 19 [Epub ahead of print].
Genetic Variation in Nicotine Metabolism Predicts the Efficacy of Extended-duration Transdermal Nicotine Therapy
CYP2A6 is the primary metabolizer of nicotine to cotinine, and cotinine to 3'-hydroxycotinine. Genetic variants have been identified that cause reduced-activity and inactive variants of the CYP2A6 gene associated with slower nicotine clearance. A phenotypic assay that measures the plasma 3'-hydroxycotinin/cotinine nicotine metabolite ratio (NMR) levels in smokers is used as a biomarker to predict the efficacy of transdermal nicotine. In the study presented here, Dr. Lerman and her colleagues tested the hypothesis that the reduced metabolizer subgroup, defined by either CYP2A6 genotype or NMR, would be more likely to benefit from a 6-month therapy with transdermal nicotine than from the standard 8 week therapy, as compared with normal metabolizers of nicotine. The results show that smokers with reduced nicotine metabolism benefit more than normal metabolizers from extended (6-month) transdermal nicotine therapy as compared to standard therapy. The group-by-treatment interaction was significant only for the genotype and NMR measure for reduced metabolizers but not the normal metabolizers. The benefit, however, dissipates once treatment ends, suggesting that the benefits may be enhanced with longer treatment. These results suggest that determining the NMR or genotyping CYP2A6 can be used to tailor the type, dose, and length of smoking cessation treatment in clinical practice. Lerman C, Jepson C, Wileyto EP, Patterson F, Schnoll R, Mroziewicz M, Benowitz N, and Tyndale RF. Genetic variation in nicotine metabolism predicts the efficacy of extended-duration transdermal nicotine therapy. Clin Pharm Ther. 2010 Mar 24 [epub ahead of print].
Interaction of the Mu-opioid Receptor with GPR177 (Wntless) Inhibits Wnt Secretion: Potential Implications for Opioid Dependence
Opiate agonists such as morphine and heroin exert their actions by activating the mu opioid receptor. Chronic use of opiate agonists can lead to addiction and to tolerance. Chronic use is associated with morphological changes in neurons. Now, Dr. Jin and his colleagues have identified an orphan G coupled receptor called GPR177, the mammalian ortholog of Drosophila Wntless/Evi/Sprinter that associates with the mu opiate receptor that regulates the secretion of the wnt protein. Wnts play a significant role in pattern formation during development, regulate dendritic arborizaton, and regulate neurogenesis in hippocampus. Jin and his colleagues show that antibodies selective for the mu opioid receptor precipitate the mu opioid receptor/GPR177 complex from a rat pheochromocytoma cell line and from rat brain. Experiments using immunoelectron microscopy suggest that the mu opioid receptor and the GPR177 are co-localized in the same neurons in the striatum. Treatment of HEK293 cells that express both the mu opioid receptor and GPR177 with morphine leads to the formation GPR177-mu opioid receptor complex at the cell surface, resulting in suppression of wnt release. The suppression of wnt release could be overcome by either overexpressing GPR177 or by blocking the effects of morphine with either CTAP or naloxone, both opiate antagonists. Jin and his colleagues suggest that the suppression of the wnt could explain the loss of the dendritic tree seen with chronic morphine. Jin also suggests that morphine, which delays internalization of the mu opiate receptor suppresses the release of wnts by trapping the GPR177 receptor at the cell surface. GPR177 unable to internalize to the Golgi, required for wnt release, prevents secretion. These findings suggest a novel role for GPR177 in mediating opiate dependence. Jin J, Kittanakom S, Wong V, Reyes BA, Van Bockstaele EJ, Stagljar I, Berrettini W, Levenson R. BMC Neurosci. Interaction of the mu-opioid receptor with GPR177 (Wntless) inhibits Wnt secretion: potential implications for opioid dependence BMC Neurosci. 2010 Mar 9; 11: 33.
A Major QTL on Chromosome 11 Influences Psychostimulant and Opioid Sensitivity in Mice
C57Bl/6 mouse strain shows significantly greater locomotor response to methamphetamine and to the opiate agonist, fentanyl, than does the AJ strain of mouse. Bryant and his colleagues mapped this trait to chromosome 11 using a genetic strategy that substitutes chromosome 11 from the C57BL/6 strain of mouse with chromosome 11 from the AJ strain of mouse. To further map the genetic loci on chr 11 responsible for enhanced locomotor response to methamphetamine a B6 x (AJ chromosome 11 substitution) F2 intercross was conducted. In other words of B6 mice carrying 1 substituted AJ chromosome 11 were mated with B6 mice to produce the F1 generation. These mice have one AJ chromosome 11 and one B6 chromosome. Brother-sister matings of the B6 x AJ chromosome 11 substitution is then conducted. As a result of this mating there is random recombination between homologous chromosomes. By looking at which part of chromosome 11 is B6 or AJ across the different progeny, the trait can be better localized on the chromosome. The response to methamphetamine mapped to 40 to 60 centimorgans on chromosome 11. Examining how the genes in this region vary with a function in a given strain, a large number of genes are identified. Bryant and his colleagues suggest based on anatomical localization in the striatum that Cacna1g, which encodes the alpha 1 G subunit of the t-type calcium channel CaV3.1t, Cacng5, which encodes the voltage-dependent calcium channel subunit, syntaxin binding protein 4 (Stxbp4) and sorting nexin 11 (Snx11) are of greatest interest. All are involved in the secretion of neurotransmitter from neurons. In summary, this work suggests a common locus of action for both psychostimulants and opiates and will lead to a better understanding of individual differences in response to abused drugs. Bryant CD, Chang HP, Zhang J, Wiltshire T, Tarantino LM, Palmer AA. A major QTL on chromosome 11 influences psychostimulant and opioid sensitivity in mice. Genes Brain Behav. 2009 Nov; 8(8): 795-905.
Thrombospondin May Promote Synaptogenesis through Gabapentin Receptor in CNS
Neuronal synapses are modified cell adhesions with specialized presynaptic and postsynaptic membrane structures for neuronal transmission. The cellular and molecular events that initiate various synaptogenesis are not well defined. Dr. Ben Barres, a NIDA supported researcher at Stanford University, and his group previously reported that astrocyte-derived thrombospondin is responsible for the initiation of excitatory synapse formation in CNS. They now report that a neuronal membrane receptor with known pharmacological roles may also be the receptor for thrombospondin during brain development and repair. The identified neuronal thrombospondin receptor involved in CNS synapse formation is alpha2delta-1, the receptor for the anti-epileptic and analgesic drug gabapentin. They show that the VWF-A domain of alpha2delta-1 interacts with the epidermal growth factor-like repeats common to all thrombospondins. Alpha2delta-1 overexpression increases synaptogenesis in vitro and in vivo and is required postsynaptically for thrombospondin- and astrocyte-induced synapse formation in vitro. Gabapentin antagonizes thrombospondin binding to alpha2delta-1 and powerfully inhibits excitatory synapse formation in vitro and in vivo. These findings identify alpha2delta-1 as a receptor involved in excitatory synapse formation and suggest that gabapentin may function therapeutically by blocking new synapse formation. Eroglu C, Allen NJ, Susman MW, O'Rourke NA, Park CY, Ozkan E, Chakraborty C, Mulinyawe SB, Annis DS, Huberman AD, Green EM, Lawler J, Dolmetsch R, Garcia KC, Smith SJ, Luo ZD, Rosenthal A, Mosher DF, Barres BA. Gabapentin receptor alpha2delta-1 is a neuronal thrombospondin receptor responsible for excitatory CNS synaptogenesis. Cell. 2009; 139: 380-392.
Non-coding RNA Switches Oligodendrocyte Proliferation to Differentiation during CNS Myelination
During brain development the genetic mechanisms that switch undifferentiated precursor cells of neurons and glia to differentiating and functional mature cells are not clear. This is particularly important for neuronal axon myelination which is the function of oligodendrocytes. Oligodendrocytes proliferate along the growing axons. Upon the cessation of proliferation the differentiation and myelination is initiated. It is known that the location and myelination in the CNS is regulated mainly by controlling the progression of oligodendrocyte differentiation. A NIDA researcher, Dr. Ben Barres, and his group at the Stanford University report that non-coding RNAs are required for oligodendrocytes to switch from proliferation to differentiation during CNS development. MicroRNAs (miRNAs) are small, noncoding RNA molecules generated from longer hairpin-loop RNA sequences, which are trimmed to functional 19-21 mers by successive cleavages by the obligate miRNA processing enzymes Drosha and Dicer1, then incorporated into an RNA-induced silencing complex (RISC). When a RISC-loaded miRNA recognizes a complementary sequence, it either represses RNA translation or directly promotes the degradation of the associated mRNA. To investigate the role of microRNAs in regulating oligodendrocyte differentiation and myelination, Dr. Barres utilized transgenic mice in which microRNA processing was disrupted in oligodendrocyte precursor cells by targeted deletion of Dicer1. They found that inhibition of miRNA processing disrupts normal CNS myelination and that oligodendrocyte precursor cells lacking mature miRNAs fail to differentiate normally in vitro. They further identified three miRNAs (miR-219, miR-138, and miR-338) that are induced 10-100x during oligodendrocyte differentiation; the most strongly induced of these, miR-219, is necessary and sufficient to promote the differentiation, and partially rescues differentiation defects caused by total miRNA loss. They found miR-219 directly represses the expression of PDGFRalpha, Sox6, FoxJ3, and ZFP238 proteins, all of which normally help to promote OPC proliferation. Together, these findings show that miR-219 plays a critical role in coupling differentiation to proliferation arrest in the oligodendrocyte lineage, enabling the rapid transition from proliferation to myelination. Dugas JC, Cuellar TL, Scholze A, Ason B, Ibrahim A, Emery B, Zamanian JL, Foo LC, McManus MT, Barres BA. Dicer1 and miR-219 are required for normal oligodendrocyte differentiation and myelination. Neuron. 2010; 65: 597-611.
MicroRNA Shrinks Fat Spines
The fine regulation of the number, morphology and protein composition of dendritic spines by neuronal activity constitutes a structural basis for synaptic plasticity during learning and memory. The microRNA pathway has been implicated in the regulation of synaptic protein synthesis and ultimately in dendritic spine morphogenesis, although, the particular microRNAs (miRNAs) involved are largely unknown. Siegel and colleagues demonstrate, by performing a functional screen, that a small non-coding RNA, microRNA-138, decreases the size of dendritic spines through local downregulation of acyl protein thioesterase 1 (APT1). They used microarrays to identify miRNAs that are enriched in the immediate proximity of synapses, isolating RNA from synaptosomes as a source of synaptic miRNA and comparing miRNA levels in this fraction with those in total brain extracts. They identified several miRNAs consistently enriched in the synaptic compartment. After confirming the neuronal expression and dendritic localization of candidate miRNAs, the authors set out to investigate the role of these miRNAs in regulating dendritic spine morphology, an indicator of synaptic plasticity. For this, the authors used antisense oligoribonucleotides that inhibit the action of target miRNAs. Expression of antisense oligoribonucleotides against two candidates resulted in an increase in dendritic spine volume. The physiological function of one of the two candidates, miRNA-138, is unknown in the nervous system. Using duplex RNA, the authors found that overexpression of miRNA-138 decreased spine volume without affecting other dendrite characteristics, such as spine density and dendrite branching. As expected when spine size is decreased, the authors were able to correlate the overexpression of miRNA-138 with a decrease in AMPA receptor cluster size and the miniature excitatory postsynaptic currents (mEPSCs) mediated by these receptors. The specific effect of miRNA-138 on dendritic spine size suggests a potential role in regulating synaptic plasticity rather than in regulating other characteristics of neuronal function. A key feature of this work was to identify relevant target(s) for miRNA-138. Using reporter constructs containing their 3'm untranslated region (3'UTR) after the luciferase coding region, the most striking effect of miRNA-138 overexpression was observed with the 3'UTR of the depalmitoylation enzyme APT1. Palmitoylation has only recently been implicated in the regulation of synaptic plasticity, and changes in spine structure have been linked to a dynamic balance between the addition of palmitate to, and removal from, synaptic proteins. Therefore, the authors hypothesized that APT1 may regulate spine morphology in response to neuronal activity. Other findings confirmed APT1 as a target of miRNA-138. This study by Siegel and colleagues is the first to show a direct link between miRNA and the dynamic regulation of post-translational modification of synaptic proteins leading to changes in spine morphology. Siegel G, Obernosterer G, Fiore R, Oehmen M, Bicker S, Christensen M, Khudayberdiev S, Leuschner PF, Busch CJ, Kane C, Hbel K, Dekker F, Hedberg C, Rengarajan B, Drepper C, Waldmann H, Kauppinen S, Greenberg ME, Draguhn A, Rehmsmeier M, Martinez J, Schratt GM. A functional screen implicates microRNA-138-dependent regulation of the depalmitoylation enzyme APT1 in dendritic spine morphogenesis. Nat Cell Biol. 2009 Jun; 11(6): 705-716.
Timing of Birth and Differential Gene Regulation Determines Neuronal Fates during Limbic System Development
The striatum is the inhibitory center of the telencephalon that is vital for movement and impulse control. The amygdala is a key area modulating fear, aggression and emotionality. These two important limbic system components are formed by highly diversified types of inhibitory and excitatory neurons during brain development. How and where various types of neurons are derived and how their fates are determined have not been well understood. Joshua Corbin, a NIDA researcher at the Children's National Medical Center in Washington DC, reports that a specific lineage of neural progenitors marked by Emx1 differentially contribute to excitatory and inhibitory neurons in the striatum and amygdala. Using a combination of approaches, including genetic fate mapping, cell birth dating, cell migration assays, and electrophysiology, Corbin found that cells derived from the Emx1 lineage contribute to two distinct neuronal populations in the mature basal forebrain: inhibitory medium spiny neurons in the striatum and functionally distinct subclasses of excitatory neurons in the amygdala. Their cell birth-dating studies reveal that these two populations are born at different times during early neurogenesis, with the amygdala population born before the MSNs. In the striatum, Emx1-lineage neurons represent a unique subpopulation of MSNs: they are disproportionately localized to the dorsal striatum, are found in dopamine receiving, reelin-positive patches, and are born throughout striatal neurogenesis. In addition, Corbin's data suggest that a subpopulation of these Emx1-lineage cells originate in the pallium and subsequently migrate to the developing striatum and amygdala. An intersectional fate-mapping analysis further reveals that Emx1-lineage cells that coexpress Dlx exclusively generate MSNs but do not contribute to the excitatory neurons in the amygdala. Thus, both the timing of neurogenesis and differential combinatorial gene expression appear to be key determinants of striatal versus amygdala fate decisions of Emx1-lineage cells. Cocas LA, Miyoshi G, Carney RS, Sousa VH, Hirata T, Jones KR, Fishell G, Huntsman MM, Corbin JG. Emx1-lineage progenitors differentially contribute to neural diversity in the striatum and amygdala. Journal of Neuroscience. 2009; 29:15933-15946.