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NIDA Home > Publications > Director's Reports > May, 2008 Index    

Director's Report to the National Advisory Council on Drug Abuse - May, 2008

Research Findings - Basic Neuroscience Research

NIDA Funded Researchers Identify Gene Variant Linking Nicotine Addiction and Lung Cancer

Scientists have identified a genetic variant that not only makes smokers more susceptible to nicotine addiction but also increases their risk of developing two smoking-related diseases, lung cancer and peripheral arterial disease. The variant is closely linked to two of the known subunits of nicotine receptors (alpha3 and alpha5), the sites on the surface of many cells in the brain, lungs, and body that can be bound by nicotine. When nicotine attaches to these receptors in the brain, there are changes in cell activity that result in its addictive effects. Activation of nicotinic receptors in the lung may cause cell proliferation. The study, published in the April 3, 2008 issue of the journal Nature1, highlights the advances that are being made in genetics research using the method of genome wide association, which can now identify many gene variants that increase the risk of complex bio-behavioral disorders. Carriers of this genetic variant are more likely than non-carriers to be heavy smokers, dependent on nicotine, and less likely to quit smoking. While the variant does not increase the likelihood that a person will start smoking, it increases the likelihood of addiction for those who do smoke. The study funded by NIDA and the European Union was carried out by deCODE Genetics, a biopharmaceutical company based in Reykjavik, Iceland. The same variant was identified as one that increased risk for lung cancer in two articles appearing in the April 3rd, 2008, issues of Nature2 and Nature Genetics3, and partially funded by two other NIH institutes - the National Cancer Institute and the National Human Genome Research Institute. Previous work by scientists at the Washington University and Perlegen Biosciences led by Dr. Laura Bierut4,5, the University of Pennsylvania led by Dr. Wade Berrettini6, University of Colorado, Boulder by Dr. Isabel Schlaepfer7 all funded by NIDA also have shown independently that the alpha-5/alpha-3 nicotinic receptor subunit alleles increase the risk for heavy smoking. Future research will determine whether the association of the alpha3/alpha5 gene variant with lung cancer is due entirely to its effect on the increased quantity of cigarettes smoked or is a direct effect of nicotine on lung tissue to promote tumor growth in the lungs. 1. Thorgeirsson, T.E., Geller, F., Sulem, P., Rafnar, T., Wiste, A., et al. Variant Associated with Nicotine Dependence, Lung Cancer and Peripheral Arterial Disease. Nature, 452(7187), pp. 638-642, 2008. 2. Amos, C.I., Wu, X., Broderick, P., et al. Genome-Wide Association Scan of Tag SNPs Identifies a Susceptibility Locus for Lung Cancer at 15q25.1. Nature Genetics, Apr 2, 2008 , epub ahead of print. 3. Hung, R.J., McKay, J.D., Gaborieau, V., Boffetta, P., Hashibe, M., Zaridze, D., et al.. A Susceptibility Locus for Lung Cancer Maps to Nicotinic Acetylcholine Receptor Subunit Genes on 15q25. Nature, 452(7187), pp. 633-637, 2008. 4. Bierut, L.J., Madden, P.A.F., Breslau, N., Johnson, E.O., Hatsukami, D., Pomerleau, O.F., Swan, G.E., Rutter, J., Bertelsen, S., Fox, L., Fugman, D., Goate, A.M., Hinrichs, A.L., Konvicka, K., Martin, N.G., Montgomery, G.W., Saccone, N.L., Saccone, S.F., Wang, J.C., Chase, G.A., Rice, J.P. and Ballinger, D. Novel Genes Identified in a High-Density Genome Wide Association Study for Nicotine Dependence. Human Molecular Genetics, 16(1), pp. 24-35, 2007. 5. Saccone, S.F., Hinrichs, A.L., Saccone, N.L., Chase, G.A., Konvicka, K., Madden, P.A.F., Breslau, N., Johnson, E.O., Hatsukami, D., Pomerleau, O., Swan, G.E., et. al. Cholinergic Nicotinic Receptor Genes Implicated in a Nicotine Dependence Association Study Targeting 348 Candidate Genes with 3713 SNPs. Human Molecular Genetics, 16(1), pp. 36-49, 2007. 6. Berrettini, W., Yuan, X., Tozzi, F., Song, K., Francks, C., Chilcoat, H., Waterworth, D., Muglia, P. and Mooser, V. _-5/_-3 Nicotinic Receptor Subunit Alleles Increase Risk for Heavy Smoking. Molecular Psychiatry, 13, pp. 368-373, 2008. 7. Schlaepfer, I.R., Hoft, N.R., Collins, A.C., Corley, R.P., Hewitt, J.K., Hopfer, C.J., Lessem, J.M., McQueen, M.B., Rhee, S.H. and Ehringer, M.A. The CHRNA5/A3/B4 Gene Cluster Variability as an Important Determinant of Early Alcohol and Tobacco Initiation in Young Adults. Biological Psychiatry, epub ahead of print.

DRD1 Associated with Nicotine Dependence

Genes in the dopaminergic system mediate the reinforcing and dependence producing properties of nicotine. In this study, Dr. Huang and his colleagues from the Li laboratory examined single-nucleotide polymorphisms (SNPs) within or near the dopamine D1 receptor gene (DRD1) for their association with nicotine dependence (ND), which was assessed by smoking quantity (SQ), the Heaviness of Smoking Index (HSI), and the Fagerstroem Test for ND (FTND). The samples were obtained from 2,037 participants representing 200 European American (EA) and 402 African American (AA) families. They found significant associations with rs686 in the AA sample and of rs686 and rs4532 in the pooled sample after correcting for multiple testing. Haplotype-based association analysis revealed that haplotype C-T-A, formed by rs265973, rs265975, and rs686, was significantly associated with all three ND measures in both the AA and the pooled sample. The haplotype T-A-T, formed by rs265975, rs686, and rs4532, showed a significant association with FTND in the pooled sample. Using a luciferase reporter assay, rs686, located in the 3 untranslated region, caused differential luciferase activities, indicating that rs686 is a functional polymorphism affecting expression of DRD1. Huang, W., Ma, J.Z., Payne, T.J., Beuten, J., Dupont, R.T., Li, M.D. Significant Association of DRD1 with Nicotine Dependence. Human Genetics, 123, pp. 133-140, 2008.

Salvinorin A derivatives

Salvinorin A is a diterpene acetate ester, first isolated from the leaves of a Mexican sage in the 1980s. It has generated scientific interest for its hallucinogenic drug abuse properties, and because of its particular binding selectivity as an agonist at the kappa opioid receptor (KOR), without measurably affecting other receptors, including the mu, delta, and serotonin receptors. Its precise positioning and orientation within the transmembrane helices of the kappa receptor are still under study, and may involve induced conformational change in one of the helices upon binding, which does not take place in the mu or delta receptors, even though the three opioid receptors have a fairly conserved set of amino acid residues in the cavity where ligands bind. Interest in kappa agonists as potential analgesic therapeutics has acknowledged the existence of certain side effects, including depressive-like effects and diuresis. In the case of salvinorin A, analgesic testing results in rodents suggest a short time period (ten-fifteen minutes) to reach optimal effect, and subsiding in twenty-thirty minutes, perhaps due to enzymatic hydrolysis to the more stable Salvinorin B alcohol at the two position in the molecule. Salvinorin B has not yet been positively identified in primates as an in-vivo metabolite of Salvinorin A, but is a reasonable possibility. In order to test the idea that alcohol or ether modifications at position two in Salvinorin A may produce more stable species, and possibly partial kappa agonists, or even antagonists, a number of such derivatives have been examined by several research groups. In a collaboration between Dr. David Lee and Dr. Lee-Yuan Liu Chen, the properties of 2-methoxymethyl Salvinorin B (2-MOM Salv B) have recently been reported The binding of this compound to CHO cell membranes containing tagged KOR was three-fold greater than that of Salvinorin A, and it produced full agonism in the GTPgamma S functional assay. By measuring the loss of cell surface fluorescence, 2-MOM Salv. B was seventy-fold more effective than Salv. A in promoting internalization of the KOR. It also induced down regulation of the KOR more efficiently than did Salvinorin A, after four hours of exposure, as measured by loss of the Western immunoblot band representing the fully glycosylated tagged KOR. In terms of its in-vivo effects, 2-MOM Salv B produced persistent antinociception (ninety to one hundred twenty minutes) in rats (hot plate testing) at 2.5 and 5 mg/kg ip injection doses, and hypothermia at a dose of 1 mg/kg thirty-sixty minutes after ip injection, while Salvinorin A did not show antinociception or hypothermia at 10 mg/kg. In mice, 2-MOM Salv. B caused immobility at .05-1 mg/kg persisting three hours, a more pronounced effect than seen in rats at higher doses. The antagonist nor-BNI was able to block the antinociceptive, hypothermic, and motor effects of 2-MOM Salv. B. The synthetic modification of Salvinorin A at the two position by introduction of short chain ether substituents has been shown to regulate the properties of the KOR in both in-vitro and in-vivo animal test systems. Wang, Y., Chen, Y., Xu, W., Lee, D.Y.W., Ma, Z., Rawls, S.M., Cowan, A., and Liu-Chen, L.-Y. 2-Methoxymethyl-Salvinorin B Is a Potent _ Opioid Receptor Agonist with Longer Lasting Action in Vivo Than Salvinorin A. The Journal of Pharmacology and Experimental Therapeutics, 324(3), pp. 1073-1083, 2008.

A Comprehensive Profile of Brain Enzymes that Hydrolyze the Endocannabinoid 2-Arachidonoylglycerol: Endogenous Ligands for Cannabinoid Receptors ("Endocannabinoids") Include the Lipid Transmitters Anandamide and 2-Arachidonoylglycerol (2-AG)

Endocannabinoids modulate a diverse set of physiological processes and are tightly regulated by enzymatic biosynthesis and degradation. Termination of anandamide signaling by fatty acid amide hydrolase (FAAH) is well characterized, but less is known about the inactivation of 2-AG, which can be hydrolyzed by multiple enzymes in vitro, including FAAH and monoacylglycerol lipase (MAGL). Dr. Cravatt and colleagues have taken a functional proteomic approach to comprehensively map 2-AG hydrolases in the mouse brain. Their data reveal that approximately 85% of brain 2-AG hydrolase activity can be ascribed to MAGL, and that the remaining 15% is mostly catalyzed by two uncharacterized enzymes, ABHD6 and ABHD12. Interestingly, MAGL, ABHD6, and ABHD12 display distinct subcellular distributions, suggesting that they may control different pools of 2-AG in the nervous system. Blankman, J.l., Simon, G.M. and Cravatt, B.F. A Comprehensive Profile of Brain Enzymes that Hydrolyze the Endocannabinoid 2-Arachidonoylglycerol. Chemistry & Biology, 14(12), pp. 1347-1356, 2007.

A Practical Synthesis of the Kappa Opioid Receptor Selective Agonist (+)-5R,7S,8S-N-Methyl-N-[7-(1-pyrrolidinyl)-1-oxospiro[4,5]dec-8-yl]
benzeneacetamide (U69,593)

Morphine has been the treatment of choice for relief of severe pain for decades, but its side-effect profile, including respiratory depression and addiction, prompted researchers to seek alternative remedies. One of the approaches was to develop receptor subtype selective analgesics, reasoning that eliminating the action at multiple receptors might also eliminate unwanted side-effects. Szmuszkovicz (1999) reported the synthesis of arylacetamides such as U50, 488, spiradoline, and U69, 593 as a class of opioid agonists selective for the kappa opioid receptor. In animal studies, these compounds showed potent analgesic effects. However they exhibited unacceptable side effect profiles such as hallucinations and psychotomimesis. While these compounds were not a suitable replacement for morphine, they provided valuable research tools to identify the role of kappa opioid receptors in both normal and disease states. In this paper, the authors have reported a novel approach to the synthesis of the kappa-opioid receptor agonist, U69, 593 to produce higher yields than reported previously. This approach improved upon current methods by substituting stable and isolable cyclic sulfates for the unstable epoxides. It also provided access to gram quantities of the target compound and displayed excellent control of the relative stereochemistry. The absolute stereochemistry as well as biological activity of the U69, 593 was verified using X-ray crystal structure analysis and binding assays for the kappa opioid receptor. McElroy, T., Thomas, J.B., Brine, G.A., Navarro, H.A., Deschamps, J. and Carroll, F.I. A Practical Synthesis of the Kappa Opioid Receptor Selective Agonist (+)-5R, 7S, 8S-N-Methyl-N-[7-(1-pyrrolidinyl)-1-oxospiro[4,5]dec-8-yl]-benzeneacetamide (U69,593). Synthesis, 6, pp. 943-947, 2008.

Spinal _-Opioid Receptor-Bearing Neurons are Essential for Morphine-Induced Analgesia

The role of spinal cord _-opioid receptor (MOR)-expressing dorsal horn neurons in pain and morphine analgesia is not completely understood. NIDA-grantee Dr. Ronald Wiley (Vanderbilt University) and colleagues used intrathecal dermorphin-saporin (Derm-sap) to selectively destroy MOR-expressing dorsal horn neurons in rats. Derm-sap treatment attenuated the analgesic action of both intrathecal and systemic morphine in several pain models, while having no effect on baseline responses to painful stimuli. Thus, MOR-expressing dorsal horn neurons are essential for the actions of morphine on pain, but not critical for the response to pain in the absence of analgesics. This finding suggests that the analgesic actions of opioids may be modified by altering the activity of MOR-expressing dorsal horn neurons. Kline IV, R.H. and Wiley, R.G. Spinal _-Opioid Receptor-Expressing Dorsal Horn Neurons: Role in Nociception and Morphine Antinociception. Journal of Neuroscience, 28(4), pp. 904-913, 2008.

Secondary Cells in the Rostral Ventromedial Medulla Contribute to Opioid-Induced Analgesia via a Novel Pain Suppression Pathway

In the rostral ventromedial medulla (RVM), _-opioid agonists are believed to inhibit "secondary cells," which disinhibit descending neurons that inhibit pain transmission. These secondary cells are generally presumed to be inhibitory interneurons that serve only to regulate the activity of these output neurons. Dr. Mary Heinricher and colleagues have been studying the relationship of these secondary cells to output cells involved in producing analgesia. They found that opioid administration in rats caused these secondary cells to cease firing before the output neurons begin to fire. This suggests that the secondary cells do not directly modulate descending pain pathways, but rather modulate pain via a mechanism that is independent of their direct action on primary descending output neurons in the RVM. Cleary, D.R., Neurbert, M.J. and Heinricher, M.M. Are Opioid-Sensitive Neurons in the Rostral Ventromedial Medulla Inhibitory Interneurons? Neuroscience, 151(2), pp. 564-571, 2008.

Cannabinoid Agonists Produce Analgesia via a Direct Action on TRP Channels

The cannabinoid agonists WIN 55,212-2 and AM1241 produce analgesia in inflammatory pain models. However, the mechanisms responsible for cannabinoid-induced analgesia in sensory neurons are far from understood. Dr. Kenneth Hargreaves and colleagues have been studying potential cannabinoid actions at TRPV1 and TRPA1 channels in sensory neurons that are involved in producing analgesia, since these channels are importantly involved in pain transmission. The applications of WIN 55,212-2 and AM1241 inhibited the responses of sensory neurons to chemical pain-inducing irritants, capsaicin and mustard oil. Using TRPA1-specific small interfering RNA or TRPA1-deficient mice, it was shown that the TRPA1 channel is a sole target through which WIN 55,212-2 and mustard oil activate sensory neurons. In contrast, the AM1241 effect was mediated by an unknown channel. The knockdown of TRPA1 activity abolished the desensitizing effects of WIN 55,212-2 and AM1241 on capsaicin-activated currents. Furthermore, the WIN 55,212-2 or AM1241-induced analgesia of capsaicin-evoked pain was not present in TRPA1 knockout mice. Together, these findings demonstrate that cannabinoids exert at least some of their peripheral analgesic actions via TRPA1 channels located on sensory neurons. Akopian, A.N., Ruparel, N.B., Patwardhan, A., and Hargreaves, K.M. Cannabinoids Desensitize Capsaicin and Mustard Oil Responses in Sensory Neurons via TRPA1 Activation. Journal of Neuroscience, 28(5), pp. 1064-1075, 2008.

Live MR Imaging of Neuronal Transport of Mn2+ in the Hippocampal-Septal Circuit

Dr. Russell Jacobs and his colleagues have continued their studies on functional neuronal tract tracing in live mice by the use of manganese (Mn2+)-enhanced three-dimensional magnetic resonance imaging (MRI). Mn2+, a calcium analogue that enters neurons and other cells through divalent ion channels and is transported along neuronal pathways and across synapses, was injected into the right hippocampus of normal mice and in animals with a trisomic model of Down's syndrome (DS) and imaged with 11.7T high-resolution MRI at 0.5, 6, and 24 hours afterwards to examine signal intensity changes over time in the hippocampal-septal pathway. This pathway was chosen for study since it has been implicated both in normal memory functioning and as well in the cognitive impairment of Down's syndrome and thus allowed potential functional differences to be observed. The investigators describe a pattern of Mn2+-enhanced signal in vivo that correlated with the histological pattern in brains fixed with a classic neuronal tracer. They showed dynamic tract patterns of Mn2+ transport from the hippocampus to the septum over the 24 hours following injection. Co-registration of images from a cohort of animals allowed the investigators to apply statistical analysis that, somewhat unexpectedly, showed more robust transport in the DS model mice and indicated the complexity of neuronal transport. This powerful new technology, which was originally developed under the NIDA CEBRA program, shows great promise to provide new insights into normal neuronal functioning and in diseases with known or suspected transport defects. Bearer, E., Zhang, X. and Jacobs, R. Live Imaging of Neuronal Connections by Magnetic Resonance: Robust Transport in the Hippocampal-Septal Memory Circuit in a Mouse Model of Down Syndrome. NeuroImage 37, pp. 230-242, 2007.

Morphine Induces Defects in Early Response of Alveolar Macrophages to Streptococcus Pneumoniae by Modulating TLR9-NF-{kappa}B Signaling

Drug abuse is a significant risk factor for the development of community-acquired pneumonia where Streptococcus pneumoniae is one of the most common diagnoses among opiate abusers. This organism is responsible for more than 25% of all cases of pneumonia and is associated with an overall mortality rate of 23% among hospitalized patients. Researchers have used a mouse model of chronic morphine administration followed by intranasal inoculation with S. pneumoniae to understand the underlying mechanisms by which chronic morphine use impairs host innate immune response and increases susceptibility to S. pneumoniae. In this model, chronic morphine treatment delayed neutrophil recruitment, increased lung bacterial burden, and increased mortality in S. pneumoniae-infected mice. In addition, morphine inhibited transcription factor NF-kappaB activation and decreased the production of inflammatory cytokines (TNF- , IL-1, and IL-6) and chemokines (MIP-2 and KC) in both bronchoalveolar lavage fluid and lung tissue of infected mice. Additional studies in the mouse model showed that increased mortality and bacterial outgrowth observed in morphine-treated infected mice were further exaggerated following depletion of alveolar macrophages, suggesting that the effect of morphine is, at least in part, mediated via alveolar macrophages. To further understand the underlying molecular mechanisms whereby morphine impairs the activity of alveolar macrophages, researchers used an in vitro alveolar macrophages and lung epithelial cells infection model. In this model, exposure of cells to pneumococci resulted in significant release of MIP-2 from alveolar macrophages, but not from lung epithelial cells. Morphine treatment reduced MIP-2 release from pneumococci-stimulated alveolar macrophages. Furthermore, morphine treatment inhibited S. pneumoniae-induced NF-kappaB-dependent gene transcription in alveolar macrophages following 2 h of in vitro infection. S. pneumoniae infection resulted in a significant induction of NF-kappaB activity in TLR9 stably transfected HEK 293 cells (but not in TLR2 and TLR4 transfected HEK 293 cells), which was inhibited by morphine. Moreover, morphine treatment also decreased bacterial uptake and killing by alveolar macrophages. Taken together, these results suggest that morphine treatment impairs TLR9-NF-kappaB signaling in alveolar macrophages and subsequently diminishes bacterial clearance during the early stages of infection, leading to a compromised innate immune response. Wang, J., Barke, R.A., Charboneau, R., Schwendener, R. and Roy, S. Morphine Induces Defects in Early Response of Alveolar Macrophages to Streptococcus pneumoniae by Modulating TLR9-NF-_B Signaling. Journal of Immunology, 180(5), pp. 3594-3600, 2008.

Action Potential-Independent and Nicotinic Receptor-Mediated Transmission via Miniature Postsynaptic Currents Occurs at Hippocampal CA3-Mossy Fiber Synapses

Transmitter release across synapses is largely evoked by incoming trains of presynaptic action potentials. Although action potential-independent, spontaneous transmitter release can also be measured (termed miniature excitatory or inhibitory postsynaptic currents), the randomness and small quantal amounts of transmitter released through this mechanism have resulted in the assumption that these miniature events may not be functionally significant. This paper now shows that in hippocampal mossy fiber boutons, activation of 7-subtype nicotinic acetylcholine receptors (7-nAChR) results in a large increase in the amplitude of spontaneous release, indicating an increased concerted release of multiple quanta from the fibers. The effects of 7-nAChR activation are mediated by biologically-relevant doses of nicotine. Increased spontaneous release results from 7-nAChR-promoted increase in internal calcium levels and activation of presynaptic calcium/calmodulin-dependent protein kinase II (CaMKII). These results demonstrate a novel form of synaptic plasticity mediated by presynaptic 7-nAChR that may play a role in inducing an alternate homeostatic state in the brain that potentially contributes to nicotine addiction. Sharma, G., Grybko, M. and Vijayaraghavan, S. The Journal of Neuroscience, 28(10), pp. 2563-2575, 2008.

Dopamine and Corticotropin-Releasing Factor Synergistically Alter Basolateral Amygdala-to-Medial Prefrontal Cortex Synaptic Transmission: Functional Switch after Chronic Cocaine Administration

Basolateral amygdala (BLA) neurons provide a major excitatory input to medial prefrontal cortex (mPFC)-layer V pyramidal neurons. As the amygdala encodes the emotional intensity of environmental stimuli and outcome-action associations, altered BLA-to-mPFC synaptic transmission could lead to defective emotional information processing and decision making within the mPFC that may result in misguided and inappropriate behaviors. Recent research by Gallagher and colleagues determined the effect of chronic cocaine on excitatory transmission within the BLA-mPFC pathway regulated by dopamine and corticotropin-releasing factor (CRF). In naive animals, activation of D(1/5) receptors depressed BLA-mPFC glutamatergic transmission (EPSCs), whereas CRF1 receptor activation alone had no effect. However, if activation of D(1/5) and CRF1 receptors occurred together, there was an enhanced, synergistic depression of glutamatergic transmission to levels greater than through D(1/5) receptor activation alone. After chronic cocaine administration, the function of DA(1/5) and CRF receptors switched from inhibitory to excitatory. In cocaine-treated animals, BLA-mPFC EPSCs were reduced compared to untreated animals. But now, activation of either D(1/5) or CRF2 receptors increased the cocaine-induced, depressed EPSCs. Additionally, simultaneous activation of presynaptic D(1/5) and CRF2 receptors led to further enhancement of cocaine-suppressed excitatory transmission. These data indicate that CRF acting synergistically with DA normally potentiates D(1/5)-induced synaptic depression. However, after chronic cocaine, the combined synergistic actions of DA and CRF switch polarity to enhance facilitation of BLA-mPFC glutamatergic transmission. These functional changes may underlie the altered, possibly aberrant, decision-making process induced by chronic cocaine. Orozco-Cabal, L., Liu, J., Pollandt, S., Schmidt, K. Shinnick-Gallagher, P. and Gallagher, J.P. Dopamine and Corticotropin-Releasing Factor Synergistically Alter Basolateral Amygdala-to-Medial Prefrontal Cortex Synaptic Transmission: Functional Switch after Chronic Cocaine Administration. The Journal of Neuroscience, 28(2), pp. 529-542, 2008.

Cannabinoids Restore Activity-Driven Synapse Loss Between Hippocampal Neurons

Dendritic pruning and loss of synaptic contacts are early events in many neurodegenerative diseases. These effects are dynamic and appear to differ mechanistically from the cell death process. Cannabinoids modulate synaptic activity and afford protection in some neurotoxicity models. Recent research by Kim et al. examined the effects of cannabinoids on activity-induced changes in the number of synapses between rat hippocampal neurons in culture. Morphology and synapses were visualized by confocal imaging of neurons expressing DsRed2 and postsynaptic density protein 95 fused to enhanced green fluorescent protein (PSD95-GFP). Reducing the extracellular Mg2+ concentration to 0.1 mM for 4 hr. induced intense synaptic activity that decreased the number of PSD95-GFP puncta by 45 +/- 13 %. Synapse loss was an early event, required activation of NMDA receptors and was mediated by the ubiquitin-proteasome pathway. Full and partial cannabinoid receptor agonists inhibited PSD loss in a manner reversed by the CB1 receptor antagonist rimonabant. The protection was mimicked by inhibition of presynaptic Ca2+ channels, and the full agonist WIN55,212-2 did not prevent PSD loss elicited by direct application of glutamate, suggesting a presynaptic mechanism. Prolonged exposure to WIN55,212-2, but not the partial agonist THC, desensitized the protective effect. Treating cells that had undergone PSD loss with WIN55,212-2 reversed the loss and enabled recovery of a full complement of synapses. The modulation of synaptic number by acute and prolonged exposure to cannabinoids may account for some of the effects of these drugs on the plasticity, survival and function of neural networks. Kim, H.J., Waataja, J.J. and Thayer, S. Cannabinoids Inhibit Network-Driven Synapse Loss Between Hippocampal Neurons in Culture. Journal of Pharmacology and Experimental Therapeutics, epub ahead of print.

Opioid Receptor Subtype Expression Patterns Differ in HIV tat-exposed Glial Cell Populations: Implications for HIV- and Opioid-Induced Neuropathogenesis

Recent research suggests that substance abuse, including opiate use, may increase the risk or severity of HIV-1-associated neurological impairment, whereas glial cells and infiltrating macrophages are the predominant targets of HIV infection in the brain. Opioid analgesia is also thought to involve glial signaling, and there is a question as to whether pain management in HIV patients would exacerbate disease. To explore the role of opioids and glial signaling in HIV-induced neuronal dysfunction, and to complement their findings in animal models, a team of NIDA-supported investigators characterized expression of mu, delta and kappa opioid receptors on cultured microglia and astrocytes in the absence or presence of low doses of morphine and/or HIV Tat. Morphine treatment caused significantly decreased cell surface expression of opioid receptors in microglia but not in astrocytes. However, morphine treatment in the presence of Tat significantly increased intracellular expression of opioid receptors and prevented morphine-induced cell surface opioid receptor down-regulation in microglia. These findings show that cell surface opioid receptor expression is regulated by morphine differently in microglia and astrocytes, and that HIV-Tat could increase opioid receptor signaling in microglia by increasing receptor expression and/or altering ligand-induced trafficking of opioid receptors. The significance of these findings is that opiate exposure could either predispose HIV-infected individuals to CNS infection by suppressing microglial immune responses, or precipitate neurological abnormalities by increasing viral burden in the CNS. Turchan-Cholewo, J., Dimayuga, F.O., Ding, Q., Keller, J.N., Hauser, K.F., Knapp, P.E. and Bruce-Keller, A.J. Cell-Specific Actions of HIV-Tat and Morphine on Opioid Receptor Expression in Glia. Journal of Neuroscience Research, epub ahead of print.

Methamphetamine Inhibits Innate Immunity Against HCV Infection

There is little known about the interactions between hepatitis C virus (HCV) and methamphetamine, which is a highly abused psychostimulant and a known risk factor for human immunodeficiency virus (HIV)/HCV coinfection. A recent study by NIDA-supported investigators examined whether methamphetamine has the ability to inhibit innate immunity in the host cells, facilitating HCV replication in human hepatocytes. Methamphetamine inhibited intracellular interferon alpha expression in human hepatocytes, which was associated with the increase in HCV replication. In addition, methamphetamine also compromised the anti-HCV effect of recombinant interferon alpha. Methamphetamine also inhibited the expression of the signal transducer and activator of transcription 1, a key modulator in interferon-mediated immune and biological responses, and down-regulated the expression of interferon regulatory factor-5, a crucial transcriptional factor that activates the interferon pathway. These in vitro findings that methamphetamine compromises interferon alpha-mediated innate immunity against HCV infection indicates that methamphetamine exposure may be a cofactor in the immunopathogenesis of HCV disease. Ye, L., Peng, J.S., Wang, X., Wang, Y.J., Luo, G.X. and Ho, W.Z. Methamphetamine Enhances Hepatitis C Virus Replication in Human Hepatocytes. Journal of Viral Hepatitis, 15, pp. 261-270, 2008.

SynCAMs Organize Synapses Through Heterophilic Adhesion

Synapses are asymmetric cell junctions with precisely juxtaposed presynaptic and postsynaptic sides. Trans-synaptic adhesion complexes are thought to organize developing synapses. The molecular composition of these complexes, however, remains incompletely understood, precluding us from understanding how adhesion across the synaptic cleft guides synapse development. Dr. Thomas Biederer and colleagues define two immunoglobulin super-family members, SynCAM 1 and 2, that are expressed in neurons in the developing brain and localize to excitatory and inhibitory synapses. They function as cell adhesion molecules and assemble with each other across the synaptic cleft into a specific, transsynaptic SynCAM 1/2 complex. Additionally, SynCAM 1 and 2 promote functional synapses as they increase the number of active presynaptic terminals and enhance excitatory neurotransmission. The interaction of SynCAM 1 and 2 is affected by glycosylation, indicating regulation of this adhesion complex by posttranslational modification. The SynCAM 1/2 complex is representative of the highly defined adhesive patterns of this protein family, the four members of which are expressed in neurons in divergent expression profiles. SynCAMs 1, 2, and 3 each can bind themselves, yet preferentially assemble into specific, heterophilic complexes as shown for the synaptic SynCAM 1/2 interaction and a second complex comprising SynCAM 3 and 4. Their results define SynCAM proteins as components of novel heterophilic transsynaptic adhesion complexes that set up asymmetric interactions, with SynCAM proteins contributing to synapse organization and function. Fogel, A.I., Akins, M.R., Krupp, A.J., Stagi, M., Stein, V. and Biederer, T. SynCAMs Organize Synapses through Heterophilic Adhesion. Journal of Neuroscience, 27(46), pp. 12516-12530, 2008.

Methamphetamine Facilitates Vesicular Accumulation of Glutamate in Corticostriatal Glutamatergic Terminals

Dr. Bryan Yamamoto's group was first to show that excess glutamate (GLU) released in the striatum plays a critical role in methamphetamine (METH) neurotoxicity to dopaminergic neuron terminals, probably via glutamatergic excitotoxicity. Their recent studies provide evidence of how striatal glutamatergic transmission is increased by METH. The uptake and storage of GLU in synaptic vesicles is a primary step in glutamatergic neurotransmission. The proteins responsible for GLU uptake by vesicles in corticostriatal glutamatergic neurons are the vesicular GLU transporter-1 (VGLUT1) and the vesicle-associated glyceraldehyde-3-phosphate dehydrogenase (GAPDH)/3-phosphoglycerate kinase complex that generates the ATP (which translates into energy) required for uptake by VGLUT1. However, there was no information linking VGLUT1 with METH. The Yamamoto group now reports that METH increases cortical VGLUT1 mRNA, striatal VGLUT1 protein in sub-cellular fractions, and the Vmax (maximal rate of GLU uptake) of striatal vesicular GLU uptake. METH also increases GAPDH protein in the crude vesicle fraction. METH-induced increases in cortical VGLUT1 mRNA, as well as striatal VGLUT1 and GAPDH, are GABA(A) receptor-dependent because they are blocked by GABA(A) receptor antagonism in the substantia nigra. These results show that VGLUT1 can be dynamically regulated via a polysynaptic pathway to facilitate vesicular accumulation of GLU for subsequent release after METH. Specifically, based on earlier work of the Yamamoto group, it is evident that METH-induced increases in striatonigral GABA release act via nigral GABA(A) receptors that in turn decrease GABAergic nigrothalamic activity, disinhibit thalamocortical activity, and increase corticostriatal GLU release. The time course of enhanced VGLUT1 function parallels the protracted increases in extracellular GLU within the striatum. The underlying transcriptional events mediating the VGLUT1 and GAPDH sensitivities to METH have yet to be determined. Mark, K.A., Quinton, M.S., Russek, S.J., Yamamoto, B.K. Dynamic Changes in Vesicular Glutamate Transporter 1 Function and Expression Related to Methamphetamine-Induced Glutamate Release. Journal of Neuroscience, 27(25), pp. 6823-6831, 2007.

Nicotine, Immunity and Disease

Nicotine acts through two cholinergic receptors: nicotinic and muscarinic. A recent finding points to the involvement of both muscarinic receptors (mAChRs) and nicotinic receptors (nAChRs) on lymphocytes. Therefore, it is likely that these cholinergic receptors play a role in regulating immune responses. Indeed, activation of nAChRs inhibits adaptive and innate immune responses. However, very little is known about the role of mAChRs in the regulation of immune and inflammatory responses. Chronic nicotine administration has been demonstrated to increase the lung burden of influenza virus and Cryptococcus neoformans; two very prominent diseases associated with AIDS. As acetylcholine can react with both nicotinic and muscarinic receptors, it is difficult to ascertain whether the muscarinic (mAChR) and/or nicotinic (nAChR) receptors are driving different actions. In a recent study using selective muscarinic agonists/antagonists, Dr. Sopori's group showed that while mAChR agonists (oxotremorine) stimulate, mAChR antagonists (atropine) inhibit immune and inflammatory responses. Thus, activation of nicotinic and muscarinic receptors has opposite effects on the immune/inflammatory responses. Razani-Boroujerdi, S., Behl, M., Hahn, F.F, Pena-Philippides, J.C., Hutt, J. and Sopori, M.L. Role of Muscarinic Receptors in the Regulation of Immune and Inflammatory Responses. Journal of Neuroimmunology, 194, pp. 83-88, 2008.

Persistent Alterations in Mesolimbic Gene Expression with Abstinence from Cocaine Self-Administration

Addiction is a chronic relapsing disorder characterized by compulsive drug seeking behavior in the face of adverse consequences. This raises the question of what factors mediate persistent changes in the brain following abstinence that increase the probability of relapse. NIDA funded investigators at Pennsylvania State University now show persistent changes in mesolimbic gene expression that lasts longer than 100 days following 10 days of chronic cocaine administration in rats. At 100 days the expression of fos, arc, egr1, and Nr4a1 in the nucleus accumbens and prefrontal cortex were all significantly reduced when compared to controls. Future research will determine whether these decreases in gene expression are causally related to relapse and whether changes in chromatin remodeling affect these changes. Chromatin remodeling refers to enzymatic modification of histone proteins that surround DNA on a chromosome. Enzymatic modifications of histones are thought to determine which genes are expressed or repressed. Also, it will be of interest to determine whether persistent changes of gene expression seen in the periphery can predict relapse. Freeman, W.M., Patel, K.M., Brucklacher, R.M., Lull, M.E., Erwin, M., Morgan, D., Roberts, D.C.S. and Vrana, K.E. Persistent Alterations in Mesolimbic Gene Expression with Abstinence from Cocaine Self-Administration. Neuropsychopharmacology, 2007, epub ahead of print.

Homer Proteins: An Unanticipated Role in the Immune System

Homer proteins are cytoplasmic scaffolds with important functions at the excitatory synapses of some neurons. Dr. Worley and colleagues have previously shown that mouse mutants in Homer2 have increased behavioral sensitivity to cocaine. Although the neuronal functions of Homer proteins are becoming better understood, Homers have a wide tissue distribution including expression in the immune system. In order to study the neuronal functions of Homer proteins in living animals, Dr. Worley generated mice with deletions in each of the Homer subtypes. Interestingly mice with a Homer3 deletion had abnormal lymph nodes suggesting a potential defect in immune function. To further investigate this, Dr. Worley grew T cells (from the immune system) that lacked the Homer2 and Homer3 subtypes. Dr. Worley and co-workers found that Homer2 and 3 negatively regulate T cell activation. A variety of experimental strategies was then used to elucidate the molecular basis of this negative regulation. It was found that Homer2 and 3 could directly bind to specific NFAT transcription factor subtypes that regulate Interleukin-2 expression in T cells. Normally calcineurin, a calcium-stimulated protein phosphatase, binds the NFAT transcription factor in an identical location leading to NFAT activation. However Homer binding can compete with calcineurin binding, reducing NFAT function. Interestingly Homer3 binds NFAT through a special EVH1 protein domain and Dr. Worley found that this domain can be phosphorylated by the AKT1 protein kinase, which reduces Homer binding to NFAT. Thus Homer proteins can serve as structural as well as molecular scaffolds that are capable of integrating some of the multiple signaling pathways that regulate Interleukin-2 expression and T cell function. The serendipitous observation of an immune cell phenotype in the Homer mutant mice allowed Dr. Worley and colleagues to characterize the Homer/NFAT/calcineurin/AKT1 signaling pathways in great detail in immune cells. For the future, researchers will likely turn their attention back to the nervous system to see if these Homer regulated pathways function similarly in neurons. Huang, G.N., Huso, D.L., Bouyain, S., Tu, J., McCorkell, K.A., May, M.J., Zhu, Y., Lutz, M., Collins, S., Dehoff, M., Kang, S., Whartenby, K., Powell, J., Leahy, D. and Worley, P.F. NFAT Binding and Regulation of T Cell Activation by the Cytoplasmic Scaffolding Homer Proteins. Science, 319 (5862), pp. 476-481, 2008.

BDNF/Trk Activity Is Essential for Cocaine Responses in Adult and Increased Neuroplasticity in Postweanling Mice

It has been reported that drug-induced conditioned place preference increases with age. The molecular mechanisms underlying the increased psychostimulant effect with increased age are not fully understood. A group of NIDA researchers at Temple University, led by Dr. Ellen Unterwald, decided to look into the changing cell and molecular activities of the learning and memory circuitries by comparing the postweanling, periadolescent, and adult male CD-1 mice exposed to cocaine (20 mg/kg). The rewarding effects of cocaine were assessed, as were the response to a Trk antagonist and the regulation of dopamine and cAMP-regulated phosphoprotein, 32 kDa (DARPP-32). DARPP-32 protein has been previously found to be specifically required for drug-induced place preference. The team observed that cocaine was rewarding in both periadolescent and adult mice using a conditioned place preference procedure. In contrast, postweanling mice failed to demonstrate significant cocaine-induced place preference. Because neurotrophins, including brain-derived neurotrophic factor (BDNF) and TrkB are developmentally regulated, their role in the age specific effects of cocaine was determined using the Trk receptor antagonist K252a. Their results suggest that in postweanling mice activation of Trk receptors, likely by endogenous BDNF, reduced the rewarding effects of cocaine, because the pretreatment of mice with Trk receptor antagonist K252a enhanced the development of cocaine place preference. Furthermore, repeated cocaine was associated with increased DARPP-32 protein in postweanlings and DARPP-32 induction was prevented with K252a. These results indicate an association between BDNF, cocaine reward, and DARPP-32 in postweanling mice. Meanwhile, Dr. Unterwald points out that although inhibition of Trk clearly enhanced the acquisition of cocaine place preference and diminished the upregulation of DARPP-32, these data do not prove a direct cause and effect relationship between DARPP-32 levels and the absence or presence of a rewarding effect of cocaine. The team plans to study further whether postweanling mice are more or less sensitive to the rewarding properties of cocaine. Niculescu, M., Perrine, S.A., Miller, J.S., Ehrlich, M.E., Ellen, M. and Unterwald E.M. Trk: A Neuromodulator of Age-Specific Behavioral and Neurochemical Responses to Cocaine in Mice. Journal of Neuroscience, 28, pp. 1198-1207, 2008.

Regulation of CB1 Cannabinoid Receptor Trafficking by the Adaptor Protein AP-3

G-Protein coupled receptors (GPCRs) are generally thought of as cell surface receptors, although they are not always cell surface receptors;GPR30 as an example. But for GPCRs that function at the cell surface, it is generally thought that they need to be at the cell surface to be functional. The cannabinoid receptor 1 (CB1) is a GPCR found both at the cell surface and in intracellular vesicles. Dr. Devi recently reported that the majority of the endogenous CB1 receptors do not reach the cell surface. How and why these receptors traffic to the vesicles has been unknown and was the focus of the present study. Rozenfeld and Devi found that intracellular endogenous CB1 receptors do not have an endocytic origin. Their data suggest that CB1 receptors traffic directly to lysosomes rather than trafficking first to the cell surface and then trafficking to the lysosomes by endocytosis. They found that these receptors associate with the adaptor protein AP-3 and follow an AP-3-dependent trafficking from the biosynthetic compartment to the lysosomal compartment. If they reduced the expression of AP-3 by knocking down the delta subunit (AP-3) they observed enhanced cell surface localization of CB1 receptors. Finally, they showed that CB1 receptors in the late endosomal/lysosomal compartment are associated with heterotrimeric G proteins and appear to mediate signal transduction. These results suggest that intracellular CB1 receptors may be functional and that their spatial segregation may significantly affect receptor function. Rozenfeld, R. and Devi, L.A. Regulation of CB1 Cannabinoid Receptor Trafficking by the Adaptor Protein AP-3. The FASEB Journal, epub ahead of print March 7, 2008.


Research Findings

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