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Either your web browser doesn't support Javascript or it is currently turned off. In the latter case, please turn on Javascript support in your web browser and reload this page. Refer to the copyright information in the article for licensing details. Free full text in Europe PMC. Agonism of the glucagon-like peptide 1 GLP-1 receptor GLP-1R has been effective at treating aspects of addictive behavior for a number of abused substances, including cocaine. However, the molecular mechanisms and brain circuits underlying the therapeutic effects of GLP-1R signaling on cocaine actions remain elusive. Recent evidence has revealed that endogenous signaling at the GLP-1R within the forebrain lateral septum LS acts to reduce cocaine-induced locomotion and cocaine conditioned place preference, both considered dopamine DA -associated behaviors. Interestingly, acute administration of Ex-4 reduces septal expression of the retrograde messenger 2-arachidonylglycerol 2-AG , as well as a product of its presynaptic degradation, arachidonic acid AA. Cocaine addiction is a highly prevalent disorder characterized by continued drug use in spite of negative consequences. Despite the harmful effects of cocaine, the development of pharmacotherapies to treat cocaine addiction has been slow. This is because modulators of addictive behavior are limited and we lack a comprehensive understanding of their mechanisms. Starting with the discovery that glucagon-like peptide 1 GLP-1 regulates amphetamine-induced locomotion, 1 GLP-1 receptor GLP-1R agonism has been shown to reduce the rewarding properties of cocaine and other drugs of abuse in rodents. GLP-1 is both a hormone produced by L cells of the intestine and a neuropeptide produced by the nucleus of the tractus solitarius. The neurotransmitter dopamine DA underlies the rewarding properties of drugs of abuse, as well as natural rewards. One such node is found within the forebrain lateral septum LS. Recently, Harasta et al. This phenomenon is associated with increased DAT surface expression and function mediated by a reduction in endocannabinoid and arachidonic acid AA levels in the LS. These findings reveal a novel mechanism of GLP-1R action in brain and provide a more comprehensive understanding of how emerging GLP-1R-targeted therapies affect signaling in the brain. All the experiments were performed during the light cycle. For immunohistochemical characterization of GLP-1R localization, bacterial artificial chromosome transgenic mice containing a fluorescent reporter expressed in GLP-1R-expressing cells were generated as described in Supplementary Information. The suppliers for all chemicals not otherwise identified can be found in the Supplementary Information. The sections were immunostained and imaged as described in the Supplementary Information. The procedure for c-fos IHC is as previously described. Microdialysis was performed as described previously 4 with few modifications. The dialysis probe was positioned in LS, anteroposterior: 0. The first two min samples were discarded to obtain stable basal values. Hereafter, two min fractions were collected to establish baseline DA levels. Subsequently, the mice were injected with Ex-4 or vehicle, and three min fractions were collected. All the fractions were assayed immediately after collection using high-performance liquid chromatography with electrochemical detection. Total protein was taken, and the samples were processed for protein concentration using a Bio-Rad Philadelphia, PA, USA protein assay and spectrometry at nm. Equivalent volumes of the sample were added to 3 ml of scintillation fluid Ecoscint H, National Diagnostics, Atlanta, GA, USA and radioactive counts were measured by scintillation counter. The mice were injected with Ex-4 or vehicle intraperitoneally 30 min before being killed. The mice were rapidly decapitated and the brains were obtained and blocked to obtain DS and LS punches. The samples were homogenized in methanol and centrifuged; water was added to the supernatant for a final ratio of methanol:water. The pellets were resuspended and processed for protein concentration using a Bio-Rad protein assay and spectrometry at nm. Krebs—Henseleit buffer was used m m NaCl, 4. The cell viability was measured using Trypan blue staining. Immunoprecipitations were performed using 1 mg total protein. The data were examined with Student's t -test for comparisons of only two data sets and one-way or two-way analysis of variance for comparisons of multiple groups, followed by Tukey's- or Bonferroni-corrected comparisons. Optical density of bands was determined using Image J software. The statistical analysis was performed with GraphPad Prism software, version 5. The methods for Supplementary Figures can be found in the Supplementary Information. To this end, we generated a transgenic mouse expressing a bacterial artificial chromosome for the protein mApple under the control of the promoter for the GLP-1R. The mApple signal was amplified by immunostaining Figures 1a, c, d and f and DAT was labeled in parallel Figures 1b, c, e and f. Within the caudal LS Figures 1d—f , there is similar evidence for this anatomical overlap. The DA terminals are labeled as two strips extending dorsomedially to ventrolaterally in the LSi. The GLP-1R-expressing cells are also found in the caudal LSi, but the cell bodies additionally extend further into the more medial laterodorsal tegmental area. Dotted lines delineate region boundaries. Dotted box includes the LS. Extracellular concentrations of DA are expressed as the percentage of basal levels in two fractions collected before the intervention; significant analysis of variance. We next tested whether a therapeutically relevant administration that is, systemic administration of Ex-4 was capable of altering neuronal activity in the LS. We first evaluated the effect of Ex-4 on the activation of septal neurons by measuring c-fos expression. Ex-4 administration intraperitoneally 30 min before killing decreased basal c-fos gene expression by in situ hybridization relative to vehicle-treated mice Figures 1h and i. This result indicates that systemic administration of Ex-4 alters neuronal activity in the LS. The psychogenic and addictive properties of cocaine are mediated, at least in part, through the blockade of DAT-mediated DA reuptake and an increase in extracellular DA. We determined the effect of systemic Ex-4 on local cocaine-induced changes in DA homeostasis in vivo within the LS, as measured by microdialysis in freely moving mice. Systemic pretreatment with Ex-4 intraperitoneally 90 min prior impaired the cocaine's ability to significantly increase extracellular DA. Ex-4 or vehicle was perfused through the microdialysis probe concurrently with cocaine Supplementary Figure 2B. The local administration of Ex-4 impaired the ability of cocaine to increase extracellular DA levels. These studies were performed in slices in which only the local circuits are intact; therefore, we can assume that our observations were the result of local GLP-1R stimulation and not the result of feed-forward circuit-level changes. The slices were treated with 1 n m GLP-1 for 20 min. Indeed, 2-AG hydrolysis via monoacylglycerol lipase is the primary mechanism for free AA generation in the central nervous system. To this end, mice were injected with Ex-4 intraperitoneally 30 min prior to sacrifice, and the LS was punched and analyzed by mass spectrometry for 2-AG and free AA levels. Significant analysis of variance followed by post hoc tests. The PC12 cells contain vesicles capable of neurotransmitter release. Notably, psychostimulants are known to induce oxidative stress, 55 , 56 , 57 whereas GLP-1R signaling, which reduces AA levels, is known to reduce it. These results show that in the presence of AA, there is a significant reduction in DAT function under both oxidizing and non-oxidizing conditions. Notably, a greater loss of function was observed under oxidizing conditions, which also affects DAT's apparent affinity for DA Figure 3f. DAT plasma membrane trafficking is a key regulator of DA transport capacity. Our results indicate that in hDAT cells, AA, under both oxidative and non-oxidative conditions, decreases significantly and to the same extent plasma membrane DAT Figures 4a and b as determined by biotinylation. These data also point to additional mechanisms promoted in terms of DAT function by oxidative stress. Lipid rafts are regions highly enriched in phospholipids containing AA, and DAT localization to lipid rafts has been associated with regulation of DAT function and trafficking. In the presence of salicylamine, there were no significant changes in Vmax Figure 5g or Km Figure 5h when cells were treated with AA under non-oxidative or oxidative conditions with respect to the control condition. Importantly, our findings demonstrate that DAT is a target of AA oxidative products, and that this interaction has profound functional implications. Synthetic isoketals form adducts with human dopamine transporter hDAT and decrease its function. No significant differences were observed between the groups. The statistical analysis was performed with analysis of variance. AA, arachidonic acid; Veh, vehicle. Since the discovery that the LS is a potent site of electrical self-stimulation in both rodents and humans, 20 , 71 it has historically received relatively little attention as a reward center. Recent studies, however, suggest that it integrates output from traditional reward areas including the VTA and lateral hypothalamus. GLP-1Rs are highly expressed in the LS and their signaling has been shown to attenuate the locomotor response to the psychostimulant d -amphetamine, 1 to reduce cocaine CPP, 14 , 16 and to block self-administration for cocaine in rodents. Cocaine, by targeting the DAT, increases extracellular DA levels, which leads to cocaine's behavioral actions. DAT expression level is an important factor in determining the effects of cocaine, likely through effects on basal DA tone. For example, extended access to cocaine, which results in escalating cocaine intake, causes lower expression of striatal DAT and reduced DA clearance in rats. However, it is clear to us that other mechanisms in addition to DAT occupancy participate in the behavioral actions of cocaine. These kinds of mechanisms have been defined for other drugs of abuse, such as morphine, and may be contributing in parallel to altered cocaine reward. This increase translates into an increase in transport capacity, and is paralleled by reduced ability of cocaine to increase extracellular DA levels. Our data suggest that increasing the amount of DAT expressed at the cell surface may reduce the extent to which cocaine is effective at increasing extracellular DA within the LS. These data offer an additional mechanism for GLP-1R signaling regulation of the reinforcing and rewarding properties of cocaine without precluding the possibility that GLP-1R signaling may also affect DA release and firing properties of VTA neurons. Here we find that stimulating the GLP-1R through administration of Ex-4 in a therapeutically relevant manner systemic administration reduces septal 2-AG levels. Indeed, 2-AG diffuses across synapses. Of note, AA is known to facilitate a pro-inflammatory state in the brain; 86 GLP-1 analogs, on the other hand, possess both anti-inflammatory and neuroprotective effects. On the basis of our findings, GLP-1R signaling may oppose the effects of psychostimulants by affecting levels of AA as well as oxidative stress. We believe that the increase in DAT surface expression promoted by GLP-1R signaling represents a mechanism by which this neuropeptide controls both cocaine-induced increase in LS DA as well as cocaine actions. The treatment of hDAT cells with AA under conditions favoring lipid peroxidation significantly decreased DAT surface expression and transport capacity; the latter effect was also demonstrated with synthetic isoketals. These results are consistent with the known ability of isoketals to impair the function of plasma membrane proteins. However, this reduction was smaller than that obtained under conditions favoring lipid peroxidation or in the presence of isoketals. These results provide a mechanism for the results reported by Chen et al. However, we hypothesize that covalent interactions of AA metabolites with specific lysine residues on the DAT support these actions. This decrease is important as cocaine has been shown to increase 2-AG tone in the brain. These brain regions include the nucleus accumbens and VTA, which have been identified as targets of Ex-4 actions on brain reward. Our study points to septal GLP-1R as a potential novel target for the treatment of drug abuse. SP has an active research contract with Lundbeck Pharmaceuticals; however, the work presented in the manuscript was supported entirely by the grants named above and Vanderbilt University. The remaining authors declare no conflict of interest. Sci Rep , 14 1 , 18 Oct Cited by: 0 articles PMID: Int J Mol Sci , 25 5 , 05 Mar Front Psychiatry , , 29 Aug Jerlhag E. Front Pharmacol , , 30 Mar Exp Clin Psychopharmacol , 31 3 , 08 Dec This data has been provided by curated databases and other sources that have cited the article. To arrive at the top five similar articles we use a word-weighted algorithm to compare words from the Title and Abstract of each citation. Neurochem Int , , 25 May Physiol Behav , , 11 Jun Neuropsychopharmacology , 40 8 , 11 Feb Life Sci , 73 6 , 01 Jun Cited by: 31 articles PMID: Europe PMC requires Javascript to function effectively. Search life-sciences literature 44,, articles, preprints and more Search Advanced search. This website requires cookies, and the limited processing of your personal data in order to function. By using the site you are agreeing to this as outlined in our privacy notice and cookie policy. Reddy IA 1 ,. Pino JA 2, 8 ,. Weikop P 3 ,. Osses N 2 ,. Bering T 3 ,. Valle C 4 ,. Bluett RJ 1, 7 ,. Erreger K 5 ,. Wortwein G 3 ,. Reyes JG 2 ,. Graham D 6 ,. Stanwood GD 6 ,. Hackett TA 1 ,. Patel S 7 ,. Fink-Jensen A 3 ,. Torres GE 8 ,. Galli A 1, 5. Affiliations 1. Authors Valle C 4. Authors Erreger K 5 Galli A 1, 5. Share this article Share with email Share with twitter Share with linkedin Share with facebook. Abstract Agonism of the glucagon-like peptide 1 GLP-1 receptor GLP-1R has been effective at treating aspects of addictive behavior for a number of abused substances, including cocaine. Free full text. Transl Psychiatry. Published online May PMID: Author information Article notes Copyright and License information Disclaimer. E-mail: ude. This work is licensed under a Creative Commons Attribution 4. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. This article has been cited by other articles in PMC. Go to:. Supplementary Information. Drugs and materials The suppliers for all chemicals not otherwise identified can be found in the Supplementary Information. In vivo microdialysis in mice Microdialysis was performed as described previously 4 with few modifications. Immunoprecipitations Immunoprecipitations were performed using 1 mg total protein. Open in a separate window. Figure 1. Systemic administration of a GLP-1R agonist reduces cocaine-induced DA elevation and c-fos expression in the LS We next tested whether a therapeutically relevant administration that is, systemic administration of Ex-4 was capable of altering neuronal activity in the LS. Figure 2. Figure 3. Figure 4. Figure 5. Supplementary Information Click here for additional data file. Exendin-4 decreases amphetamine-induced locomotor activity. Physiol Behav ; : — Moving beyond energy homeostasis: new roles for glucagon-like peptide-1 in food and drug reward. Neurochem Int ; 73 : 49— Septal glucagon-like peptide 1 receptor expression determines suppression of cocaine-induced behavior. Neuropsychopharmacology ; 40 : — The glucagon-like peptide 1 GLP-1 receptor agonist exendin-4 reduces cocaine self-administration in mice. Gastroenterology ; : — GLP-1 neurons in the nucleus of the solitary tract project directly to the ventral tegmental area and nucleus accumbens to control for food intake. Endocrinology ; : — The incretin system: glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors in type 2 diabetes. Lancet ; : — A randomized, controlled trial of 3. N Engl J Med ; : 11— Entry of exendin-4 into brain is rapid but may be limited at high doses. Distribution of pre-pro-glucagon and glucagon-like peptide-1 receptor messenger RNAs in the rat central nervous system. J Comp Neurol ; : — The glucagon-like peptide 1 GLP-1 analogue, exendin-4, decreases the rewarding value of food: a new role for mesolimbic GLP-1 receptors. J Neurosci ; 32 : — Gut peptide GLP-1 and its analogue, exendin-4, decrease alcohol intake and reward. PLoS One ; 8 : e The glucagon-like peptide 1 analogue exendin-4 attenuates the nicotine-induced locomotor stimulation, accumbal dopamine release, conditioned place preference as well as the expression of locomotor sensitization in mice. The glucagon-like peptide 1 analogue, exendin-4, attenuates the rewarding properties of psychostimulant drugs in mice. The glucagon-like peptide 1 analogue Exendin-4 attenuates alcohol mediated behaviors in rodents. Psychoneuroendocrinology ; 38 : — GLP-1 analog attenuates cocaine reward. Mol Psychiatry ; 18 : — The glucagon-like peptide-1 receptor as a potential treatment target in alcohol use disorder: evidence from human genetic association studies and a mouse model of alcohol dependence. Transl Psychiatry ; 5 : e How can drug addiction help us understand obesity? Nat Neurosci ; 8 : — What is the role of dopamine in reward: hedonic impact, reward learning, or incentive salience? Brain Res Brain Res Rev ; 28 : — Positive reinforcement produced by electrical stimulation of septal area and other regions of rat brain. J Comp Physiol Psychol ; 47 : — Regulation of affect by the lateral septum: implications for neuropsychiatry. Brain Res Brain Res Rev ; 46 : 71— Linking context with reward: a functional circuit from hippocampal CA3 to ventral tegmental area. Science ; : — A septal-hypothalamic pathway drives orexin neurons, which is necessary for conditioned cocaine preference. J Neurosci ; 35 : — Neuropeptide Y Y5 receptor antagonism attenuates cocaine-induced effects in mice. Psychopharmacology Berl ; : — The Mouse Brain in Stereotaxic Coordinates. J Neurosci Methods ; : 59— Insulin reveals Akt signaling as a novel regulator of norepinephrine transporter trafficking and norepinephrine homeostasis. J Neurosci ; 30 : — Genetic disruption of 2-arachidonoylglycerol synthesis reveals a key role for endocannabinoid signaling in anxiety modulation. Cell Rep ; 9 : — Ligand-induced internalization of the p75 neurotrophin receptor: a slow route to the signaling endosome. J Neurosci ; 23 : — Physical and functional interaction between the dopamine transporter and the synaptic vesicle protein synaptogyrin J Neurosci ; 29 : — The dopamine transporter: immunochemical characterization and localization in brain. J Neurosci ; 15 3 Pt 1 : — The dopamine hypothesis of the reinforcing properties of cocaine. Trends Neurosci ; 14 : — Abolished cocaine reward in mice with a cocaine-insensitive dopamine transporter. Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter. Nature ; : — Impaired striatal Akt signaling disrupts dopamine homeostasis and increases feeding. PLoS One ; 6 : 1— Hypoinsulinemia regulates amphetamine-induced reverse transport of dopamine. PLoS Biol ; 5 : — Rescue of dopamine transporter function in hypoinsulinemic rats by a D2 receptor-ERK-dependent mechanism. Rapid regulation of dopamine transporter function by substrates, blockers and presynaptic receptor ligands. Eur J Pharmacol ; : — Rapid regulation of the dopamine transporter: role in stimulant addiction? Neuropharmacology ; 47 : 80— Mice expressing markedly reduced striatal dopamine transporters exhibit increased locomotor activity, dopamine uptake turnover rate, and cocaine responsiveness. Synapse ; 67 : — Cocaine reward and locomotion stimulation in mice with reduced dopamine transporter expression. BMC Neurosci ; 8 : Exendin-4 is a high potency agonist and truncated exendin- -amide an antagonist at the glucagon-like peptide 1- -amide receptor of insulin-secreting beta-cells. J Biol Chem ; : — Reduced neural response to reward following 7 days treatment with the cannabinoid CB1 antagonist rimonabant in healthy volunteers. Int J Neuropsychopharmacol ; 13 : — Insulin induces long-term depression of ventral tegmental area dopamine neurons via endocannabinoids. Nat Neurosci ; 16 : — Endocannabinoid signalling in reward and addiction. Nat Rev Neurosci ; 16 : — Brain cannabinoid CB 2 receptors modulate cocaine's actions in mice. Nat Neurosci ; 14 : — Cannabinoid CB1 receptor antagonists attenuate cocaine's rewarding effects: experiments with self-administration and brain-stimulation reward in rats. Neuropsychopharmacology ; 33 : — Endocannabinoid hydrolysis generates brain prostaglandins that promote neuroinflammation. Effects of arachidonic acid on dopamine synthesis, spontaneous release, and uptake in striatal synaptosomes from the rat. J Neurochem ; 64 : — Inhibition by arachidonic acid and other fatty acids of dopamine uptake at the human dopamine transporter. Eur J Pharmacol ; : 89— Regulation of the functional activity of the human dopamine transporter by the arachidonic acid pathway. The PC12 cell as model for neurosecretion. Acta Physiol Oxf ; : — Role of oxidative metabolites of cocaine in toxicity and addiction: oxidative stress and electron transfer. Med Hypotheses ; 64 : — Cocaine-induced oxidative stress precedes cell death in human neuronal progenitor cells. Neurochem Int ; 50 : 69— Oxidative stress in psychiatric disorders: evidence base and therapeutic implications. Int J Neuropsychopharmacol ; 11 : — Exendin-4, a glucagon-like peptide-1 receptor agonist, provides neuroprotection in mice transient focal cerebral ischemia. J Cereb Blood Flow Metab ; 31 : — Identification of extremely reactive gamma-ketoaldehydes isolevuglandins as products of the isoprostane pathway and characterization of their lysyl protein adducts. Nat Protoc ; 2 : — Isoketals: highly reactive gamma-ketoaldehydes formed from the H2-isoprostane pathway. Chem Phys Lipids ; : 85— Pyridoxamine analogues scavenge lipid-derived gamma-ketoaldehydes and protect against H2O2-mediated cytotoxicity. Biochemistry ; 45 : — Neurochem Int ; 33 : — Peroxynitrite inactivates the human dopamine transporter by modification of cysteine potential mechanism of neurotoxicity in dopamine neurons. J Neurosci ; 22 : — Hydrogen peroxide induces loss of dopamine transporter activity: a calcium-dependent oxidative mechanism. J Neurochem ; 86 : — A closer look at amphetamine-induced reverse transport and trafficking of the dopamine and norepinephrine transporters. Mol Neurobiol ; 39 : 73— Dynamic regulation of the dopamine transporter. Biochemistry ; 41 : — Dopamine transporter endocytic trafficking in striatal dopaminergic neurons: differential dependence on dynamin and the actin cytoskeleton. J Neurosci ; 33 : — Electrical self-stimulation of the brain in man. Am J Psychiatry ; : — Brain stimulation reward and dopamine terminal fields. Septal and cortical projections. Brain Res ; : — On lateral septum-like characteristics of outputs from the accumbal hedonic 'hotspot' of Pecina and Berridge with commentary on the transitional nature of basal forebrain 'boundaries'. J Comp Neurol ; : 50— Transition from moderate to excessive drug intake: change in hedonic set point. Extended access of cocaine self-administration results in tolerance to the dopamine-elevating and locomotor-stimulating effects of cocaine. J Neurochem ; : — Cocaine self-administration produces pharmacodynamic tolerance: differential effects on the potency of dopamine transporter blockers, releasers, and methylphenidate. Neuropsychopharmacology ; 37 : — Methylphenidate and cocaine self-administration produce distinct dopamine terminal alterations. Addict Biol ; 19 : — A single amphetamine infusion reverses deficits in dopamine nerve-terminal function caused by a history of cocaine self-administration. Substrates and inhibitors display different sensitivity to expression level of the dopamine transporter in heterologously expressing cells. Relationship between subjective effects of cocaine and dopamine transporter occupancy. Role for mTOR signaling and neuronal activity in morphine-induced adaptations in ventral tegmental area dopamine neurons. Neuron ; 72 : — The glucagon-like peptides: a double-edged therapeutic sword? Trends Pharmacol Sci ; 24 : — Cyclic AMP-mediated pleiotropic effects of glucagon-like peptide-1 receptor activation. Focus on 'Exendin-4 attenuates high glucose-induced cardiomyocyte apoptosis via inhibition of endoplasmic reticulum stress and activation of SERCA2a'. The role of endocannabinoid signaling in motor control. Physiology Bethesda ; 25 : — D-Amphetamine stimulates D2 dopamine receptor-mediated brain signaling involving arachidonic acid in unanesthetized rats. J Cereb Blood Flow Metab ; 26 : — Modulation of inflammation in brain: a matter of fat. Impairment of synaptic plasticity and memory formation in GLP-1 receptor KO mice: Interaction between type 2 diabetes and Alzheimer's disease. Behav Brain Res ; : — Central effects of GLP new opportunities for treatments of neurodegenerative diseases. J Endocrinol ; : T31—T New roles for insulin-like hormones in neuronal signalling and protection: new hopes for novel treatments of Alzheimer's disease? Neurobiol Aging ; 31 : — Glucagon-like peptide 1 receptor stimulation reverses key deficits in distinct rodent models of Parkinson's disease. J Neuroinflammation ; 5 : Modification of proteins by isoketal-containing oxidized phospholipids. Cocaine self-administration differentially modulates the expression of endogenous cannabinoid system-related proteins in the hippocampus of Lewis vs. Fischer rats. Int J Neuropsychopharmacol ; 16 : — J Neurosci ; 34 : — Smart citations by scite. The number of the statements may be higher than the number of citations provided by EuropePMC if one paper cites another multiple times or lower if scite has not yet processed some of the citing articles. Explore citation contexts and check if this article has been supported or disputed. The risk of depression, anxiety, and suicidal behavior in patients with obesity on glucagon like peptide-1 receptor agonist therapy. Case Report: Semaglutide-associated depression: a report of two cases. The therapeutic potential of glucagon-like peptide-1 for persons with addictions based on findings from preclinical and clinical studies. Glucagon-like peptide-1 receptors in nucleus accumbens, ventral hippocampus, and lateral septum reduce alcohol reinforcement in mice. Data Data that cites the article This data has been provided by curated databases and other sources that have cited the article. Proteins in UniProt 8. Similar Articles To arrive at the top five similar articles we use a word-weighted algorithm to compare words from the Title and Abstract of each citation. Glucagon-like peptide-1 receptor regulation of basal dopamine transporter activity is species-dependent. Substance P and cholecystokinin regulate neurochemical responses to cocaine and methamphetamine in the striatum. Funding Funders who supported this work. External link. Please review our privacy policy.

Genomic and structural basis for evolution of tropane alkaloid biosynthesis

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Official websites use. Share sensitive information only on official, secure websites. To whom correspondence may be addressed. Email: huangjp Plants produce structurally diverse and lineage-specific natural products through a coordination of serial enzymes to catalyze sequential reactions. Cocaine and hyoscyamine with similar tropane skeletons are distributed among different families. The independent recruitments of serial biosynthetic enzymes responsible for cocaine and hyoscyamine biosynthesis were revealed on the genomic and structural levels. This rare phenomenon reminds us that the discovery of biosynthetic enzymes based exclusively on enzyme homology or key conserved sites requires caution when investigating the biosynthesis of natural products with similar chemical structure in phylogenetically distant species. Keywords: tropane alkaloids, evolution, biosynthesis, genome, structural biology. The tropane alkaloids TAs cocaine and hyoscyamine have been used medicinally for thousands of years. To understand the evolutionary origins and trajectories of serial biosynthetic enzymes of TAs and especially the characteristic tropane skeletons, we generated the chromosome-level genome assemblies of cocaine-producing Erythroxylum novogranatense Erythroxylaceae, rosids clade and hyoscyamine-producing Anisodus acutangulus Solanaceae, asterids clade. Molecular docking analysis and key site mutation experiments suggested that ecgonone synthases CYP81AN15 and CYP82M3 adopt different active-site architectures to biosynthesize the same product ecgonone from the same substrate in Erythroxylaceae and Solanaceae. The combination of structural biology and comparative genomic analysis revealed that ecgonone methyltransferase, which is responsible for the biosynthesis of characteristic 2-substituted carboxymethyl group in cocaine, evolved from the tandem copies of salicylic acid methyltransferase by the mutations of critical E and S residues. Overall, we provided strong evidence for the independent origins of serial TA biosynthetic enzymes on the genomic and structural level, underlying the chemotypic convergence of TAs in phylogenetically distant species. Plants obtain structurally rich and functionally diverse natural products during their evolution 1. It is common to find natural products with similar chemical structures existing in phylogenetically close species. Beyond that, some similar natural products, such as tropane alkaloids TAs 2 , spiroketal steroids 3 , and iridoids 4 , show scattered distributions in phylogenetically distant plants. At present, our understanding of the scattered distributions of natural products is dominated by elucidating the evolution of individual biosynthetic enzyme 3 — 6. The biosynthetic pathway of natural products involves a complex coordination of a set of enzymes which catalyze sequential reactions 7. However, little is known about how a set of enzymes involved in the biosynthesis of natural products exhibiting a scattered distribution pattern evolved in plants. Gene duplication, followed by neofunctionalization, is a major contributor to the diversity of natural products in plants 8. Neofunctionalization can be obtained through a small number of key mutations, which can be identified via protein structural and functional analyses 9 , Besides obtaining the neofunctionalized gene, other mutated genetic material generated by gene duplication events can be either lost or remained These genetic traces are subsequently deposited within the plant genome and can be used to trace the ancestral state and locus of a neofunctional gene, in turn, shedding light on the evolutionary process Therefore, the combination of structural analysis and genomic analysis can provide strong evidence to further explore the evolutionary origins and trajectories of biosynthetic enzymes. Tropane alkaloids can be divided into two major groups: tropinone and ecgonone derivatives Fig. The tropinone group i. The ecgonone group, represented by cocaine, mainly exists in Erythroxylaceae family 12 , Cocaine Goprelto and Numbrino has been approved by the food and drug administration as a topical anesthetic. After a century of investigation, the complete biosynthetic pathway for hyoscyamine and scopolamine has been fully described, and their de novo production in yeast was achieved 15 , Recently, we reported a near-complete biosynthetic route of cocaine 17 , Cocaine and hyoscyamine share a similar biosynthetic pathway Fig. In contrast to the one-step catalysis of putrescine N -methyltransferase PMT to form N -methylputrescine in solanaceous species, an alternative biosynthetic route for N -methylputrescine formation was revealed in Erythroxylaceae Fig. In addition, the biosynthetic flux to tropinone or ecgonone group is controlled via En MT4-catalyzed methylation of ecgonone, thereby avoiding the spontaneous decarboxylation of unstable ecgonone to form tropinone Fig. The subsequent reduction and esterification of methylecgonone or tropinone drive the formation of cocaine or littorine the precursor of hyoscyamine Fig. Previous protein structural and phylogenetic analyses suggested that polyketide synthases in cocaine and hyoscyamine biosynthesis were independently recruited 5. These similar biosynthetic intermediates and different enzyme strategies made us curious about the evolutionary origins and trajectories of serial enzymes involved in the biosynthesis of TAs, especially the characteristic tropane skeletons i. The similar biosynthetic routes of cocaine and hyoscyamine. In this study, protein structural and functional analysis showed that ecgonone synthases CYP81AN15 and CYP82M3 in Erythroxylaceae and Solanaceae adopted different active-site architectures to biosynthesize the same product, and ecgonone methyltransferase En MT4 most likely evolved from salicylic acid methyltransferase SAMT in Erythroxylum novogranatense. Two high-quality genome assemblies of cocaine-producing E. Further comparative genomic and phylogenetic analyses were used to explore the genomic events that led to the independent emergence of a set of TA biosynthetic enzymes in Erythroxylaceae and Solanaceae. To understand the genomic contexts that underpin the emergence of TA biosynthesis in plants, the chromosome-level genomes of hyoscyamine-producing A. S1 and Table S1. The assembly quality of these two genomes was high based on benchmarking universal single-copy orthologs BUSCO analysis To better understand the evolutionary relationships between E. This analysis revealed that the possible splitting time between A. The within-species Ks synonymous substitutions per synonymous site distribution of paralogous genes in A. The peak at 0. S1 and intergenomic collinearity analysis SI Appendix , Fig. The distribution of Ks values for paralogous genes in E. Intergenomic synteny analysis between E. Subsequently, based on the estimated divergence time in the phylogenetic tree between A. Inferred phylogenetic tree based on single-copy orthologs of 11 plants. The approximate times of A. Methylation of putrescine or spermidine is the first specific step in TA biosynthesis Fig. Combined with phylogenetic analysis and intergenomic comparative analysis SI Appendix , Figs. The blue circles or triangles represent the existence of PKS gene in the selected species. A flavin-dependent amine oxidase AOF1 from erythroxylaceous species catalyzes the removal of aminopropyl group in N -methylspermidine to form N -methylputrescine Fig. Phylogenetic analysis of genome-wide samples of polyamine oxidases from core eudicot species showed that the AOF1-located branch consists only of protein sequences from the rosids SI Appendix , Fig. S9 and S The independent recruitment of PMT enzymes responsible for N -methylputrescine formation in solanaceous plants resulted in the divergence of polyamine methylation, which is the first specific step of TA biosynthesis in both rosids Erythroxylaceae and asterids Solanaceae Figs. S11 A. These results indicated ambiguities as to the origin and evolutionary history of the Solanales lineage PYKS gene. S11 B. S12 , indicating that A. S11 C. To validate the roles of these predicted residues, site-directed mutants of CYP81AN15 were generated. The catalytic activities were almost abolished by the mutation of TA or QA, and similar results were observed when the amide group in Q residue was replaced with a carboxyl group by the mutation of QE Fig. S13 , suggesting that T and Q cooperate to govern the direction of carboxy group of MPOA via salt bridge or hydrogen bond. In contrast, functionally similar substitution DE showed S13 , indicating that the carboxyl group in the D residue plays a critical role in the position control of the pyrrole ring through salt bridge interaction with the N atom of the pyrrole ring. Three biological duplications were performed for each assay. N and O atoms are colored blue and red, respectively. As a result, its catalytic activity was completely abolished by the mutation of DA Fig. S15 and Tables S6. Therefore, this tandem replication event likely took place before the divergence of eudicot plants and provided generous CYP81 family homologous genes for the evolution of EnCYP81AN Aa Contig2. S15 , did not contain the key T, Q, and D sites, while CYP82M3 was recruited to be responsible for the biosynthesis of the tropane skeleton in Solanaceae. S16 , suggesting independent recruitment of ecgonone synthase in Solanaceae. The existence of a 2-substituted carboxymethyl group in cocaine is attributed to the participation of ecgonone methyltransferase En MT4 in E. To trace the origin of En MT4, we performed intergenomic comparative genomics analysis of the chromosomal region surrounding EnMT4. As a result, there were two significant chromosomal expansion regions regions A and B in E. B Molecular docking of the ecgonone intermediate with predicted En MT4. C atoms in ecgonone and En MT4 residues are colored green and cyan, respectively. We performed molecular docking with ecgonone in the predicted protein structure of En MT4. Q25 and W were supposed to orient the carboxylate of ecgonone Fig. Q25 could form a hydrogen bond with the N atom of tropane ring Fig. S19 A. For the substrate specificity of ecgonone, the hydroxyl group of tropane ring might be positioned by hydrogen bonds with E and S Fig. Consequently, the catalytic activities of En MT4 for methylation of ecgonone were abolished by the polar-to-nonpolar EL substitution and small-to-large SY substitutions or significantly decreased by large-to-small EA and polar-to-nonpolar SA substitutions Fig. S19 B. These results indicated that E and S perform critical roles in En MT4 catalysis of methylecgonone formation. S19 C. In addition, the Ks ratios among EnMT4 and its homologous genes were mainly around 0. Thus, these E. Nevertheless, the expression of both SlMecgoR and AaMecgoR was largely undetectable in roots 23 , suggesting a minimal contribution by MecgoR to tropinone reduction in Solanaceae. In agreement with this function, we only found syntenic orthologs of AaLS in the genome of hyoscyamine-producing D. At the genome-wide level, no genes with high predicted sequence similarity to EnCS were detected in close relatives of E. Further intragenomic sequence homology investigation implied a probable mechanism by which the CS gene emerged in Erythroxylaceae Fig. The absence of high-quality genome sequences of medicinal TA-producing plants has hindered the understanding of TA biosynthesis and evolution. In this study, we generated high-quality chromosome-level genome assemblies of TA-producing E. This early differentiation point provided sufficient time for the independent evolution of cocaine and hyoscyamine biosynthesis through gene multiplication, gene loss, tandem duplication, segmental duplication, and key site mutation. The following specific polyketide synthase PYKS and ecgonone synthase CYP82M3 in Solanaceae were recruited to biosynthesize the tropane skeleton of hyoscyamine 27 , After their species divergence, ecgonone methyltransferase En MT4 evolved via Erythroxylaceae-specific segmental duplication and subsequent mutations of key E and S residues, thus stabilizing the carboxyl group of ecgonone and directing the biosynthetic flux to ecgonone derivatives i. Aside from the early tandem duplication for CYP81AN15 evolution, the recent LTR-RT-mediated duplications probably provided numerous templates for the evolution of ecgonone methyltransferase En MT4 and cocaine synthase CS , which drove the lineage-specific emergence of cocaine in Erythroxylaceae. Overall, the analysis of evolutionary origins and trajectories of tropane skeleton biosynthesis on the genomic and structural levels deepen our understanding of the occurrence of lineage-specific tropane alkaloids in Solanaceae, Erythroxylaceae, and Rhizophoraceae. In summary, a set of enzymes involved in the biosynthesis of scattered TAs potentially emerged via 1 the evolution of SPMT from SPDS through hexaploidy event in core eudicot species and the emergence of PMT from SPDS once again in the Solanales, 2 parallel recruitment of polyketide synthases and ecgonone synthases, 3 Erythroxylaceae-specific evolution of ecgonone methyltransferase which directs the biosynthetic flux to ecgonone derivatives, and 4 the lineage-specific recruitment of reductases and acyltransferases from distinct families for the postmodification of the tropane skeleton in cocaine and hyoscyamine. Young leaves of E. The genome sequencing, assembly, and annotation process were described in SI Appendix , Materials and Methods. Orthologous gene families were clustered from proteins of 11 plants from different families by OrthoFinder Protein sequences of single-copy orthologous families were aligned by MAFFT 38 and were then corrected by Gblocks To time the E. Then, we estimated E. In the same way, the recent WGD event in A. Intergenomic and intragenomic synteny analyses of genomic regions near functionally characterized genes were performed by MCScanX The products were detected at nm. Assay which lacked enzyme served as a negative control blank. The injections were eluted with a min gradient mobile phase program 0. The key parameters of algorithm and grid number were set as default. The complex structures with favorable orientation and low binding energy were selected for subsequent analysis. The interactions between ligands and proteins were visualized by PyMOL 2. The assays were carried out in triplicate, and the specific activities of mutated En MT4 were calculated based on the peak area ratios of methylecgonone between mutated and wild En MT4. The assembly mixtures were transformed into chemically competent E. The resulting leaves were harvested 1 d later and were lyophilized to determine the dry weight of the leaves. After being ground in liquid nitrogen, the pulverized leaves were extracted with CH 2 Cl 2. The obtained aqueous layers were adjusted to pH 9. The assays were carried out in triplicate, and the specific activities of mutated Ab CYP82M3 were calculated based on the peak area ratios of tropinone between mutated and wild Ab CYP82M3. We thank Prof. We thank Profs. The E. The fifteen transcriptome datasets of A. All data that support the findings of this study are available in the main text and the SI Appendix. This section collects any data citations, data availability statements, or supplementary materials included in this article. As a library, NLM provides access to scientific literature. Find articles by Yong-Jiang Wang. Find articles by Tian Tain. Find articles by Jia-Yi Yu. Find articles by Jie Li. Find articles by Bingyan Xu. Find articles by Jianghua Chen. Find articles by Jian-Ping Huang. Find articles by Sheng-Xiong Huang. Published by PNAS. Open in a new tab. Click here for additional data file. Similar articles. Add to Collections. Create a new collection. Add to an existing collection. Choose a collection Unable to load your collection due to an error Please try again. Add Cancel.

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