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Director's Report to the National Advisory Council on Drug Abuse - May, 2005

Research Findings - Basic Neurosciences Research

α5 Nicotinic Acetylcholine Receptor Subunit mRNA Levels During Brain Development

This study compared pre- and postnatal expression of the α5 nicotinic acetylcholine receptor (nAChR) subunit in rat cortex and hippocampus. Similar to expression of other nAChR subunits, there was transient expression of α5 mRNA during cortical and hippocampal development. During the first two postnatal weeks of development, transient expression was detected mainly in cortical layers V and I/II. In the hippocampus, transiently increased expression was detected in CA1 and CA3 pyramidal neurons and granule cells of the dentate gyrus; areas have low expression levels in the adult. Co-expression of α5 and α7 mRNAs was detected in a subpopulation of hippocampal interneurons. In contrast, in the subiculum, numerous cells exhibited strong hybridization signals for α5 and α7 mRNAs, but co-expression was rarely detected. The investigators concluded that this expression pattern places nicotinic receptors containing the α5 subunit in a position to regulate glutamatergic and GABAergic transmission during the postnatal period. Winzer-Serhan, U.H. and Leslie, F.M. Expression of Alpha5 Nicotinic Acetylcholine Receptor Subunit mRNA During Hippocampal and Cortical Development. Journal of Comparative Neurology, 481(1), pp. 19-30, 2005.

CB2 Cannabinoid Receptor and Mechanisms of Peripheral Analgesia

CB2 cannabinoid receptor-selective agonists are promising candidates for the treatment of pain. CB2 receptor activation inhibits acute, inflammatory, and neuropathic pain responses but does not cause central nervous system (CNS) effects, consistent with the lack of CB2 receptors in the normal CNS. To date, there has been virtually no information regarding the mechanism of CB2 receptor-mediated inhibition of pain responses. Here, Ibrahim and colleagues test the hypothesis that CB2 receptor activation stimulates release from keratinocytes of the endogenous opioid β-endorphin, which then acts at opioid receptors on primary afferent neurons to inhibit nociception. The antinociceptive effects of the CB2 receptor-selective agonist AM1241 were prevented in rats when opioid antagonist naloxone or antiserum to β-endorphin was injected in the hind paw where the noxious thermal stimulus was applied, suggesting that β-endorphin is necessary for CB2 receptor-mediated antinociception. Further, AM1241 did not inhibit nociception in μ-opioid receptor-deficient mice. Hind paw injection of β-endorphin was sufficient to produce antinociception. AM1241 stimulated β-endorphin release from rat skin tissue and from cultured human keratinocytes. This stimulation was prevented by AM630, a CB2 cannabinoid receptor-selective antagonist and was not observed in skin from CB2-cannabinoid receptor-deficient mice. These data suggest that CB2 receptor activation stimulates release from keratinocytes of β-endorphin, which acts at local neuronal mu-opioid receptors to inhibit nociception. Supporting this possibility, CB2 immunolabeling was detected on B-endorphin-containing keratinocytes in stratum granulosum throughout the epidermis of the hind paw. This mechanism allows for the local release of β-endorphin, where CB2 receptors are present, leading to anatomical specificity of opioid effects. Ibrahim, M.M., Porreca, F., Lai, J., Albrecht, P.J., Rice, F.L., Khodorova, A., Davar, G., Makriyannis, A., Vanderah, T.W., Mata, H.P. and Malan, T.P., Jr. CB2 Cannabinoid Receptor Activation Produces Antinociception by Stimulating Peripheral Release of Endogenous Opioids. Proceedings of the National Academy of Sciences, 102, pp. 3093-3098, 2005.

Receptor Affinity Ligands

The design of ligands as "reporters" of reactivity toward protein receptors, transporters, or channels has frequently been based on covalent modification of amino acids specific to these proteins. Examples of this technique include the isothiocyanate group used in opioid and cannabinoid receptor ligands, and the photo-activated azido group, used in tropane and pyridyl ligands, targeting the dopamine transporter and the nicotinic receptor, respectively. Dr. Philip Portoghese and his collaborators have pursued the approach of targeting appropriately spaced cysteine thiol and lysine amino groups with a ligand produced from naltrexamine and ortho-phthalaldehyde or ortho-naphthaldehyde. These ligands have the following properties: they are capable of labeling mu, kappa, or delta opioid receptor membranes from HEK-293 cells with high affinity, and in an irreversible (wash-resistant) manner. The labeling also produces a fluorescence in the 533-546 nm range when laser excitation of 460-490 nm is used, and the development of this fluorescence can be followed kinetically by flow cytometry. The fluorescence is characteristic of that for a benzoisoindole ring, formed by stepwise reaction of the ligand with a lysine amino group, followed by reaction of the resulting imine with a neighboring cysteine thiol group, to produce a 2-thiobenzoisoindole ring. Background or non-specific fluorescence could be minimized by the inclusion of an antagonist such as naltrexone in the analysis. In the case of the ortho-naphthaldehyde ligand, site-directed mutational analysis of the mu receptor has suggested that the lysine 233 and the cysteine 235 in transmembrane helix five are the reactive sites for this ring formation. Further experiments may include full pharmacological characterization (the cross-linked product should produce receptor antagonism) and confirmation of the reaction sites by digests of various receptor regions. Zhang, Y., McCurdy, C.R., Metzger, T.G., and Portoghese, P.S. Specific Cross-Linking of Lys233 and Cys235 in the Mu Opioid Receptor by a Reporter Affinity Label. Biochemistry, 44, pp. 2271-2275, 2005.

Preparation of Monoclonal Antibodies Reactive to the Endogenous Small Molecule, Anandamide

This report describes the hapten design and carrier molecule strategy that the authors used to generate a panel of monoclonal antibodies (mAB) to the endogenous cannabinoid, anandamide (N-arachidonylethanolamide, AEA) in order to develop an easy and inexpensive immunoassay for measuring this endogenous cannabinoid. These authors designed and successfully prepared a hapten, N-arachidonyl-7-amino-6-hydroxy-heptanoic acid (AHA), which retained the basic characteristic features of anandamide - the carboxamide, the hydroxyl and the lipophilic arachidonyl moiety with its skipped double bond system, while still showing the attachment to protein. In addition, a secondary alcohol structure was added to reduce the potential for biological hydrolysis of the hapten. Because of the diverse responses obtained after coupling this hapten to four different carriers, the authors determined that the type of carrier molecule used was particularly important for generating anti-anandamide antibodies. This report further describes the characteristics of a panel of 11 mAB, generated from four separate fusions, with a range of relative affinities and cross reactivities. Excellent selectivity for anandamide vs. two other endogenous cannabinoids and arachidonic acid was achieved following this strategy (cross reactivities <5%). In addition, at least one mAB maintained specificity for anandamide compared to two very closely related fatty acid amide molecules. However, the IC50 values in a standard enzyme-linked immunosorbent assay (ELISA) format (ca. 2-3 uM) indicated that improvement in antibody affinities or assay format will be required for an immunoassay to measure endogenous levels. Basta, P., Adcock, A.R., Tallent, C.R., Fleming, D.N., Seltzman, H.H., Whisnant, C.C., and Cook, C.E. Preparation of Monoclonal Antibodies Reactive to the Endogenous Small Molecule, Anandamide. Journal of Immunological Methods, 285, pp. 181-195, 2004.

Morphine and Stress Response in Adult Female Offspring

It has been shown that adult female rats react to stressors more intensely than adult male rats and that opioids have an inhibitory effect on the stress response. Furthermore, the response of the hypothalamic pituitary adrenal (HPA) axis to stress is known to be gender specific. In a recent paper, Dr. Ilona Vathy and her associates report that prenatal morphine exposure alters the HPA axis-regulated stress response and the sensitivity of negative feedback that are affected by the fluctuation of ovarian hormones. This study examined the effects of prenatal morphine exposure on ACTH and CORT plasma concentrations before and after restraint stress in proestrus (high estrogen level) and diestrus (low estrogen level) female rats. Prenatal morphine exposure differentially altered the ACTH and CORT responses to stress and the sensitivity of negative feedback of glucocorticoid (GR) probably by affecting the characteristics of GR receptors, and thereby altering the HP axis-controlled stress response. Slamberova, R., Rimanoczy, A., Riley, M.A., and Vathy, I. Hypothalamo-Pituitary-Adrenal Axis-Regulated Stress Response and Negative Feedback Sensitivity is Altered by Prenatal Morphine Exposure in Adult Female Rats. Neuroendocrinology, 80, pp. 192-200, 2004.

Corticotropin Releasing Factor Antagonism Reduces Cocaine-Induced DA Neuronal Activity and DA Overflow in the Nucleus Accumbens

Corticotropin releasing factor (CRF) is a neuropeptide associated with the integration of the physiological and behavioral responses to stress. More recently, CRF1 receptor antagonists have been shown to decrease cocaine self-administration and inhibit stress-induced reinstatement of cocaine-seeking behavior. The exact mechanisms underlying this effect are not clear. Based on the large literature demonstrating an association between dopaminergic neurotransmission and reward-related behavior, the aim of the present study was to examine the effects of acute vs. chronic CRF1 receptor blockade on mesencephalic dopamine (DA) neuron activity (determined by in vivo extracellular recordings) and extracellular DA levels in the nucleus accumbens (Acb) (using in vivo microdialysis). In addition, the effect of CRF1 receptor antagonism on cocaine-induced DA overflow in the Acb was examined and correlated with DA neuron activity in the ventral tegmental area (VTA). Acute (but not chronic) CRF1 receptor blockade (by CRA-0450) significantly increased DA neuron population activity without affecting burst firing, average firing rate or accumbal DA concentrations. In addition, both acute and chronic CRF1 receptor antagonism significantly reduced cocaine-stimulated DA overflow in the Acb, and this reduction was correlated with an attenuated cocaine-induced inhibition of DA population activity. Taken as a whole, these data demonstrate that, although DA neuron population activity exhibits tolerance to chronic CRF1 receptor antagonism (by CRA-0450), tolerance does not develop to the selective inhibition of cocaine-induced DA release (in the Acb) and as such may be of benefit in the treatment of cocaine addiction. Lodge, D.J., and Grace, A.A. Acute and Chronic CRF1 Receptor Blockade Inhibits Cocaine-induced Dopamine Release: Correlation with Dopamine Neuron Activity. Journal of Pharmacology and Experimental Therapeutics, (epub ahead of publication), March 22, 2005.

Relative Opioid Efficacy is Determined by the Complements of the G Protein-Coupled Receptor Desensitization Machinery

G protein-coupled receptor regulation by G protein-coupled receptor kinases and beta-arrestins can lead to desensitization and subsequent internalization of the receptor. For in vitro and cellular systems, beta-arrestins do not seem to play a major role in regulating mu opioid receptor (μOR) responsiveness. Removal of the beta arrestin2 (βarr2) gene in mice leads paradoxically to enhanced and prolonged μOR-mediated antinociception. The barr2 knockout (βarr2-KO) mice also fail to develop morphine antinociceptive tolerance in the hot-plate test, further indicating that the βarr2 protein plays an essential role in μOR regulation in vivo. In this study, the contribution of βarr2 to the regulation of the μOR was examined in both human embryonic kidney 293 cells and in βarr2-KO mice after treatment with several opiate agonists. A green fluorescent protein tagged barr2 was used to assess receptor-βarr2 interactions in living cells. Opiate agonists that induced robust βarr2-green fluorescent protein translocation produced similar analgesia profiles in wild type and βarr2-KO mice, whereas those that do not promote robust βarr2 recruitment, such as morphine and heroin, produce enhanced analgesia in vivo. In this report, Dr. Laura Bohn presents a rationale to explain the seemingly paradoxical relationship between b-arrestins and μOR regulation wherein morphine-like agonists fail to promote efficient internalization and resensitization of the receptor. Bohn, L.M., Dykstra, L.A., Lefkowitz, R.J., Caron, M.G., and Barak, L.S. Relative Opioid Efficacy is Determined by the Complements of the G Protein-Coupled Receptor Desensitization Machinery. Molecular Pharmacology, 66(1), pp. 106-112, 2004.

Microglial Activation Precedes Dopamine Terminal Pathology in Methamphetamine-Induced Neurotoxicity

Previous studies have demonstrated methamphetamine (METH)-induced toxicity to dopaminergic and serotonergic axons in rat striatum. Although several studies have identified the nature of reactive astrogliosis in this lesion model, the response of microglia has not been examined in detail. In this investigation, Dr. Hastings and her research team at the University of Pittsburgh characterized the temporal relationship of reactive microgliosis to neuropathological alterations of dopaminergic axons in striatum following exposure to methamphetamine. Adult male Sprague-Dawley rats were administered a neurotoxic regimen of methamphetamine and survived 12 h, or 1, 2, 4, and 6 days after treatment. Immunohistochemical methods were used to evaluate reactive changes in microglia throughout the brain of methamphetamine-treated rats, with a particular focus upon striatum. Pronounced morphological changes, indicative of reactive microgliosis, were evident in the brains of all methamphetamine-treated animals and were absent in saline-treated control animals. These included hyperplastic changes in cell morphology that substantially increased the size and staining intensity of reactive microglia. Quantitative analysis of reactive microglial changes in striatum demonstrated that these changes were most robust within the ventrolateral region and were maximal 2 days after methamphetamine administration. Analysis of tissue also revealed that microglial activation preceded the appearance of pathological changes in striatal dopamine fibers. Reactive microgliosis was also observed in extra-striatal regions (somatosensory and piriform cortices, and periaqueductal gray). These data demonstrate a consistent, robust, and selective activation of microglia in response to methamphetamine administration that, at least in striatum, precedes the appearance of morphological indicators of axon pathology. These observations raise the possibility that activated microglia may contribute to methamphetamine-induced neurotoxicity. LaVoie, M.J., Card, J.P. and Hastings, T.G. Microglial Activation Precedes Dopamine Terminal Pathology in Methamphetamine-Induced Neurotoxicity. Experimental Neurology, 187, pp. 47-57, 2004.

Serotonergic Neurotoxicity Enhances Methamphetamine and Cocaine Conditioned Place Preference

In mice, a neurotoxic regimen of fenfluramine, which selectively damaged their serotonergic axon terminals, enhanced methamphetamine and cocaine-induced conditioned place preference (CPP), while having no effect on lithium-induced aversive conditioning (conditioned place aversion, CPA). In the same study, selective dopaminergic neurotoxicity, induced by methamphetamine in the mice, decreased methamphetamine and cocaine CPP, and had no effect on lithium CPA. Dual dopaminergic/serotonergic neurotoxicity had no apparent effect on CPP; however, CPA was attenuated. An implication for humans is that drug-induced neurotoxicity may modulate, and even may predispose to, further drug abuse. Achat-Mendes, C., Ali, S.F., and Itzhak, Y. Differential Effects of Amphetamines-Induced Neurotoxicity on Appetitive and Aversive Pavlovian Conditioning in Mice. Neuropsychopharmacology, epub advance online publication, 2005.

Thrombospondins are Astrocyte-Secreted Proteins that Promote CNS Synaptogenesis

The proper formation of synapse and neural circuits is essential for the coordination and execution of adaptive behavior. Abnormal development of neural circuit and synaptic connection and abnormalities in the mechanisms underlying synaptic plasticity may underlie neuropsychiatric and addictive disorders. Synapses are tight junctions where a neuron releases a chemical signal, a neurotransmitter, to communicate with a neighboring neuron. Thus, a key question is how synapses form and how they are modified. Recent work by Dr. Ben Barres and his colleagues has found, surprisingly, that glia secrete factors that induce the formation of mature synapses. Mature synapses are characterized by the ability of the transmitting neuron to secrete neurotransmitter and by the ability of the receiving neuron to have receptors inserted into the membrane and clustered closely opposite of the site where neurotransmitter is being released, and the formation of tight junctions. The result that glia play an active role in synapse formation is surprising because glia were thought only to play a housekeeping role by supporting their survival and that neurons were the key players in synapse formation. In the February 11, 2005 issue of Cell, Dr. Ben Barres and his colleagues report the identification of thrombospondin 1 and 2 as some of the factors secreted by glia to promote synapse formation. Mice lacking thrombospondin 1 and thrombospondin 2 have 40 percent fewer synapses. Addition of thrombospondin 1 and 2 to neurons in culture induces the formation of synapse and induces the machinery necessary for transmitter release. However, the synapses are silent because the AMPA receptors are not inserted into the receiving neuron. Because completely functional synapses occur in the presence of glia, at least one other unidentified protein in addition to thrombospondin 1 and 2 is needed to produce a fully functional synapse. In future work, Dr. Barres hopes not only to identify the secreted factor but the receptors to which thrombospondin 1 and thrombospondin bind and induce the formation of "silent synapses." This breakthrough opens new avenues of research. Scientists can now test whether drugs of abuse modulate synapses by altering the amount or function of thrombospondins in glia. This research may also lead to the development of treatments for repair of the nervous system after injury. Christopherson, K.S., Ullian, E.M., Stokes, C.C., Mullowney, C.E., Hell, J.W., Agah, A., Lawler, J., Mosher, D.F., Bornstein, P. and Barres, B.A. Cell 120(3), pp. 421-433, February 11, 2005.

Modulation of Nicotine but not Ethanol Preference by the Mouse Chrna4 A529T Polymorphism

Alcohol and nicotine are commonly abused together. Some evidence suggests that a common set of gene variants underlies addiction to both nicotine and alcohol. Mice carrying a polymorphism of the alpha 4 subunit of the nicotinic acetylcholine receptor (AchR) in which threonine is substituted for an alanine at amino acid 527 in the alpha 4 subunit show increased consumption and preference for both alcohol and nicotine. Dr. Allan Collins and his colleagues determined whether the preference for alcohol and nicotine could be explained solely by the polymorphism in the alpha 4 subunit or could be explained by another gene in close proximity of the alpha 4-receptor gene. Dr. Collins crossed mice carrying the alanine and threonine gene variants of the alpha 4-receptor gene into mice lacking beta2 subunit of the nicotinic receptor. These mice are unable to make functional alpha4 beta2 nicotinic receptors. The increased preference for nicotine in the mice carrying the threonine variant of the alpha4 receptor lacking the beta2 subunit over mice carrying alanine variant of the alpha4 AchR lacking the beta2 is abolished but the difference in alcohol preference is not. Thus threonine substitution in the alpha4 AchR explains nicotine preference because the loss of the receptor eliminates any difference. However, because ethanol preference remains in the absence of functional alpha4 beta2 nicotinic receptors a gene lying in close proximity to the alpha4 receptor gene must exist. This is because genes lying in close proximity are less likely to recombine and segregate independently when eggs and sperm are developing during the process of meiosis. A question for future investigation is the identity of the gene variant underlying the increased preference for ethanol. Butt, C.M., King, N.M., Hutton, S.R., Collins, A.C. and Stitzel, J.A. Behavioral Neuroscience, 119(1), pp. 26-37, 2005.

Nicotinic Acetylcholine Receptor Containing the Beta4 Subunit Mediate Nicotine Withdrawal

Withdrawal from nicotine is one of the greatest obstacles that people addicted to nicotine face when they attempt to quit smoking. The actions of nicotine are mediated by a number of different types of nicotinic acetylcholine receptors in the brain. Two major types of nicotine receptors in the brain are nicotinic receptors that consist of either the alpha-4 subunit and the beta-2 subunit or the alpha-4 subunit and the beta-4 subunit. To determine which nicotine receptor is relevant to withdrawal Dr. Mary Debiasi and her colleagues at Baylor College of Medicine developed a mouse model of withdrawal. Mice were infused with nicotine using a mini-osmotic pump and withdrawal was precipitated with mecamylamine, a nicotine receptor antagonist. Withdrawal in mice is characterized by behavioral symptoms of withdrawal measured as increased grooming, chewing, scratching, shaking, jumping, and leg tremors. Mary Debiasi and her colleagues report in the November 10, 2004 issue of the Journal of Neuroscience that mutant mice lacking the beta2 subunit that are insensitive to the rewarding properties of nicotine still show behavioral signs of withdrawal while mice lacking the beta4 subunit do not show withdrawal symptoms. This result suggests that the alpha4beta4 subunit mediates withdrawal to nicotine and suggests targets for treating nicotine dependence. The results also suggest that gene variants in the beta4 subunit may affect response to nicotine in humans. To test this idea Mary Debiasi in collaboration with John Dani and Arthur Beaudet have identified four human variants of the beta4 subunit: Thr to Ile at codon 91 (T91I), Arg to Trp at codon 136 (R136W), Ser to Gly at codon 140 (S140G), and Met to Val at codon 467 (M467V). Receptors containing the R136W and M467V gene variants for the beta4 subunit were more sensitive to nicotine while T91I mutation was less sensitive to nicotine. All of these mutations showed enhanced desensitization when compared to wild type. The S140G has no effect. Future research will determine whether these gene variants are associated with differences in the severity of nicotine withdrawal. Salas, R., Pieri, F. and De Biasi, M. Journal of Neuroscience, 24(45), pp. 10035-10039, 2004; Liang, Y., Salas, R., Marubio, L., Bercovich, D., De Biasi, M., Beaudet, A.L. and Dani, J.A. Neurogenetics, 6(1), pp. 37-44, 2005.

Lmo Mutants Reveal a Novel Role for Circadian Pacemaker Neurons in Cocaine-Induced Behaviors

Fruit fly genetics provides a powerful approach to identify the molecular mechanisms underlying biological processes without any preconceived ideas. Jay Hirsh at the University of Virginia and Ulricke Heberlein at the University of California have developed genetic screens in the fruit fly drosophila melanogaster to uncover the molecular mechanism underlying the acute affects of cocaine. In the December 2004 issue of PLOS Biology Dr. Ulricke Heberlein and her colleagues describe the discovery of mutations in the LIM-only (LMO), encoding a regulator of LIM-homeodomain proteins, that alter the response to cocaine. Mutations that increase expression of LMO decrease sensitivity of flies to cocaine while decreased expression of LMO increases the sensitivity of flies to cocaine. Restoring normal levels of LMO in the PDF-expressing ventral lateral neurons (LNvs), the principal neurons that generate circadian rhythms, of mutant flies is sufficient to reestablish normal sensitivity to cocaine. As might be expected flies carrying the mutations LMO also have circadian rhythm defects. Dr. Heberlein and her colleagues were able to separate the role that (LNvs) play in regulating cocaine sensitivity from the role these neurons play in regulating circadian rhythm behavior. The response of flies to cocaine was not altered during any portion of the light dark cycles. Mutations that ablate the expression of PDF peptide in the (LNvs) affected circadian rhythms but not sensitivity to cocaine. Acute inactivation of (LNvs) neurons by transiently expressing tetanus toxin or an inwardly rectifying potassium channel increased sensitivity to cocaine without affecting circadian rhythms. This work together suggests that there is an overlap between neuronal systems that regulate circadian rhythms and cocaine sensitivity. Future studies will determine the relevance of LMO mutations in the mouse. Tsai, L.T., Bainton, R.J., Blau, J. and Heberlein, U. PLoS Biol, 2(12), e408, 2004.

Mapping of a Quantitative Trait Locus (QTL) For Morphine Withdrawal Severity

Chronic morphine exposure results in physical dependence, manifested by physical symptoms during naloxone-precipitated withdrawal. Jumping frequency is widely considered the most sensitive and reliable index of withdrawal intensity in mice. Inbred mouse strains surveyed for naloxone-precipitated withdrawal display large and significant strain differences in jumping frequency, including an approximately tenfold difference between C57BL/6 and 129P3 mice. In the present study, (B6 X 129)F2 hybrid mice were given daily morphine injections for four days using an escalating dosing schedule, and measured naloxone-precipitated withdrawal on day 5. A whole-genome scan for linkage to phenotypic data was performed using polymorphic microsatellite markers. Significant linkage was observed between withdrawal jumping frequencies and 28 cM-wide region of Chromosome 1, accounting for ~20% of the overall phenotypic variance. Two other suggestive QTLs were found on Chromosomes 5 and 10, and an additive model fitting all three loci accounted for ~43% of the total variance. The Chromosome 1 QTL for naloxone-precipitated withdrawal severity in morphine-dependent mice, which was named Depmq1, harbors approximately 100 known genes. Although QTL mapping does not indicate which gene(s) are relevant to the phenotype under study, two genes within this region are high priority candidates. These are Plcd4, encoding phospholipase C δ4 subunit (PLC) and Ugt1a1, encoding an isoform of the UDP-glucoronosyl-transferase family 1. The Depmq1 QTL is the first to be associated with naloxone-precipitated withdrawal jumping frequency, the most commonly used measure of withdrawal severity in morphine-dependent mice. Kest, B., Palmese, C.A., Juni, A., Chesler, E.J., and Mogil, J.S. Mapping of a Quantitative Trait Locus for Morphine Withdrawal Severity. Mammalian Genome, 15, pp. 610-617, 2004.

Identification of Tyrosine Hydroxylase as a Physiological Substrate for Cdk5

Kinases are enzymes that covalently bind phosphate groups to selected serines, threonines and tyrosines on proteins. The addition of the phosphate group causes dramatic changes in the substrate protein structure or function or both. For example, Dr. Bibb had previously reported that one of the substrates for cyclin-dependent kinase-5 (Cdk5) is Thr75 of the dopamine and cyclic-AMP-regulated phosphoprotein, Mr 32,000 (DARPP-32). Upon phosphorylation of Threonine 75, DARPP-32 is converted to an inhibitor of PKA, another kinase. Tyrosine hydroxylase (TH) is an enzyme involved in cellular synthesis of dopamine, one of the key molecules involved in the pleasurable effects of drugs and natural rewards. In this study, Dr. Bibb and collaborators report evidence that Cdk5 is able to phosphorylate TH directly as well as indirectly by activating another phosphorylation cascade, which ultimately phosphorylates TH. Furthermore, they identified Ser31 as the specific site at which Cdk5 is adding the phosphate group to TH. The effect of phosphorylation at Ser31 is activation of TH. Interestingly, previous studies have shown that acute administration of cocaine causes a decrease in the phosphorylation of TH Ser31. In this study authors observed increased phosphorylation of Ser31 with chronic administration. Chronic cocaine administration has also been shown to increase Cdk5 levels in the same brain regions. This study provides evidence for a mechanism by which Cdk5 may impact dopamine synthesis and furthermore that this mechanism is involved in chronic, but not acute cocaine administration. Kansy, J.W., Daubner, S.C., Nishi, A., Sotogaku, N., Lloyd, M.D., Nguyen, C., Lu, L., Haycock, J.W., Hope, B.T., Fitzpatrick, P.F. and Bibb, J.A. Journal of Neurochemistry, 91(2), pp. 374-384, 2004.

Activation of Peripheral Cannabiniod Receptors Reduces Neuronal Correlates of Pain in Rats

Andrea Hohmann and colleagues has been studying the utility of peripherally administered cannabinoid CB2 receptor agonists in reducing pain in animal models. In the current study, the authors found that peripheral application of CB2-selective cannabinoid agonist AM1241 reduced the activity of spinal wide dynamic range (WDR) fibers, which are involved in pain transmission from the spinal cord to the brain. AM1241 reduced the WDR activity that was produced by electrical stimulation of the hind paw, as well as that produced by paw inflammation. The responses of other non-pain related fibers were not reliably altered by AM1241. These findings demonstrate that the activation of CB2 receptors selectively attenuates the activity of a well-known component of a major pain pathway. These data support the potential efficacy of peripherally acting CB2 receptor agonists in the treatment of pain, an approach that would avoid many of the side effects of centrally-acting cannabinoids. Nackley, A.G., Zvonok, A.M., Makriyannis, A. and Hohmann, A.G. Activation of Cannabinoid CB2 Receptors Suppresses C-fiber Responses and Windup in Spinal Wide Dynamic Range Neurons in the Absence and Presence of Inflammation. J Neurophysiol. 92(6), pp. 3562-3574, December 2004.


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